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Sample records for acid amplification tests

  1. Automated nucleic acid amplification testing in blood banks: An additional layer of blood safety

    Pragati Chigurupati

    2015-01-01

    Full Text Available Context: A total of 30 million blood components are transfused each year in India. Blood safety thus becomes a top priority, especially with a population of around 1.23 billion and a high prevalence rate of human immunodeficiency virus (HIV, hepatitis B virus (HBV and hepatitis C virus (HCV in general population. Nucleic acid amplification testing (NAT in blood donor screening has been implemented in many developed countries to reduce the risk of transfusion-transmitted viral infections (TTIs. NAT takes care of the dynamics of window period of viruses and offers the safest blood pack for donation. Aims: The aim of this study is to show the value of NAT in blood screening. Settings and Design: Dhanavantari Blood Bank, Rajahmundry, Andhra Pradesh, India. Subjects and Methods: Over a period of 1 year from January 2012 to December 2012, a total number of 15,000 blood donor samples were subjected to tests for HIV, HBV, and HCV by enzyme-linked immunosorbent assay (ELISA method and 8000 ELISA nonreactive samples were subjected for NAT using multiplex polymerase chain reaction technology. Results: Of the 15,000 donors tested, 525 were seroreactive. In 8000 ELISA negative blood samples subjected to NAT, 4 donor samples were reactive for HBV. The NAT yield was 1 in 2000. Conclusions: NAT could detect HIV, HBV, and HCV cases in blood donor samples those were undetected by serological tests. NAT could interdict 2500 infectious donations among our approximate 5 million annual blood donations.

  2. Culture confirmation of gonococcal infection by recall of subjects found to be positive by nucleic acid amplification tests in general practice

    Møller, Jens Kjølseth

    2010-01-01

    To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community.......To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community....

  3. Tissue donation and virus safety: more nucleic acid amplification testing is needed.

    Pruss, A; Caspari, G; Krüger, D H; Blümel, J; Nübling, C M; Gürtler, L; Gerlich, W H

    2010-10-01

    In tissue and organ transplantation, it is of great importance to avoid the transmission of blood-borne viruses to the recipient. While serologic testing for anti-human immunodeficiency virus (HIV)-1 and -2, anti-hepatitis C virus (HCV), hepatitis B surface antigen (HBsAg), anti-hepatitis B core antigen (HBc), and Treponema pallidum infection is mandatory, there is until now in most countries no explicit demand for nucleic acid amplification testing (NAT) to detect HIV, hepatitis B virus (HBV), and HCV infection. After a review of reports in the literature on viral transmission events, tissue-specific issues, and manufacturing and inactivation procedures, we evaluated the significance of HIV, HCV, and HBV detection using NAT  in  donors of various types of tissues and compared our results with the experiences of blood banking organizations. There is a significant risk of HIV, HCV, and HBV transmission by musculoskeletal tissues because of their high blood content and the high donor-recipient ratio. If no effective virus inactivation procedure for musculoskeletal tissue is applied, donors should be screened using NAT  for  HIV, HCV, and HBV. Serologically screened cardiovascular tissue carries a very low risk of HIV, HCV, or HBV transmission. Nevertheless, because effective virus inactivation is impossible (retention of tissue morphology) and the donor-recipient ratio may be as high as 1:10, we concluded that NAT  should be performed for HIV, HCV, and HBV as an additional safety measure. Although cornea allografts carry the lowest risk of transmitting HIV, HCV, and HBV  owing to corneal physiology, morphology, and the epidemiology of corneal diseases, NAT  for  HCV should still be performed. If the NAT  screening of a donor for HIV, HCV, and HBV is negative, quarantine storage of the donor tissue seems dispensable. In view of numerous synergistic effects with transfusion medicine, it would be advantageous for tissue banks to cooperate with blood

  4. Detection of Chlamydia trachomatis by Nucleic Acid Amplification Testing: Our Evaluation Suggests that CDC-Recommended Approaches for Confirmatory Testing Are Ill-Advised

    Schachter, Julius; Chow, Joan M.; Howard, Holly; Bolan, Gail; Moncada, Jeanne

    2006-01-01

    We evaluated three CDC-suggested approaches for confirming positive nucleic acid amplification tests (NAATs) for Chlamydia trachomatis: (i) repeat the original test on the original specimen, (ii) retest the original specimen with a different test, and (iii) perform a different test on a duplicate specimen. For approach 1, specimens (genital swabs or first-catch urine [FCU]) initially positive by the Abbott LCx Probe System Chlamydia trachomatis Assay (LCx; Abbott Laboratories), the APTIMA Com...

  5. Nucleic acid-amplification testing for hepatitis B in cornea donors.

    Fornés, Maria Gema; Jiménez, Maria Angustias; Eisman, Marcela; Gómez Villagrán, Jose Luis; Villalba, Rafael

    2016-06-01

    Careful donor selection and implementation of tests of appropriate sensitivity and specificity are of paramount importance for minimizing the risk of transmitting infectious diseases from donors to corneal allograft recipients. Reported cases of viral transmission with corneal grafts are very unusual. Nevertheless potential virus transmission through the engraftment cannot be ruled out. According to European Guideline 2006/17/EC, screening for antibodies for Hepatitis B core antigen (anti HBc) is mandatory, and when this test is positive, some criteria must be established before using corneas. Despite the continuous progress in screening tests, donors carrying an occult hepatitis B infection (OBI) can cause transplant-transmitted hepatitis B. To date, Nucleic Acid Testing (NAT) is not an obligatory assay in corneal tissue setting neither in our country nor in the rest of European countries. Herein, we report three cornea donors that were rejected with the diagnosis of OBI through the testing of sensitive NAT and the serological profile of Hepatitis B virus. The aim of this report is to emphasize the need to include NAT in new reviews of EU Tissues and Cells Directives in order to increase level of security in tissue donation as well as not to reject a high number of donors with isolated profile of anti HBc in geographical areas with high prevalence of Hepatitis B, that could be rejected without a true criterion of Hepatitis B infection. PMID:26685699

  6. Nucleic acid amplification tests (NAATs for gonorrhoea diagnosis in women: Experience of a tertiary care hospital in north India

    Seema Sood

    2014-01-01

    Full Text Available Background & objectives: Gonorrhoea is among the most frequent of the estimated bacterial sexually transmitted infections (STIs and has significant health implications in women. The use of nucleic acid amplification tests (NAATs has been shown to provide enhanced diagnosis of gonorrhoea in female patients. However, it is recommended that an on-going assessment of the test assays should be performed to check for any probable sequence variation occurring in the targeted region. In this study, an in-house PCR targeting opa-gene of Neisseria gonorrhoeae was used in conjunction with 16S ribosomal PCR to determine the presence of gonorrhoea in female patients attending the tertiary care hospitals. Methods: Endocervical samples collected from 250 female patients with complaints of vaginal or cervical discharge or pain in lower abdomen were tested using opa and 16S ribosomal assay. The samples were also processed by conventional methods. Results: Of the 250 female patients included in the study, only one was positive by conventional methods (microscopy and culture whereas 17 patients were found to be positive based on PCR results. Interpretation & conclusions: The clinical sensitivity of conventional methods for the detection of N. gonorrhoeae in female patients was low. The gonococcal detection rates increased when molecular method was used giving 16 additional positives. Studies should be done to find out other gene targets that may be used in the screening assays to detect the presence of gonorrhoea.

  7. External Quality Assessment Program for Chlamydia trachomatis Diagnostic Testing by Nucleic Acid Amplification Assays

    Land, Sally; Tabrizi, Sepehr; Gust, Anthony; Johnson, Elizabeth; Garland, Susan; Dax, Elizabeth M.

    2002-01-01

    We report the results from 57 Australian diagnostic laboratories testing two external quality assessment panels using either the Roche Amplicor Chlamydia trachomatis test (R-PCR) or the Abbott LCx Chlamydia trachomatis assay (A-ligase chain reaction [LCR]). Panel samples were either normal urine spiked with Chlamydia trachomatis antigen or clinical urine specimens. There was no significant difference between laboratories or between assays in detection of C. trachomatis-positive clinical sampl...

  8. Electrical and Electrochemical Monitoring of Nucleic Acid Amplification

    Goda, Tatsuro; Tabata, Miyuki; Miyahara, Yuji

    2015-01-01

    Nucleic acid amplification is a gold standard technique for analyzing a tiny amount of nucleotides in molecular biology, clinical diagnostics, food safety, and environmental testing. Electrical and electrochemical monitoring of the amplification process draws attention over conventional optical methods because of the amenability toward point-of-care applications as there is a growing demand for nucleic acid sensing in situations outside the laboratory. A number of electrical and electrochemic...

  9. Comparison of an rRNA‐based and DNA‐based nucleic acid amplification test for the detection of Chlamydia trachomatis in trachoma

    Yang, Jon L; Schachter, Julius; Moncada, Jeanne; Habte, Dereje; Zerihun, Mulat; House, Jenafir I; Zhou, Zhaoxia; Hong, Kevin C; Maxey, Kathryn; Gaynor, Bruce D; Lietman, Thomas M

    2007-01-01

    Background/Aim The World Health Organisation (WHO) hopes to achieve global elimination of trachoma, still the leading cause of preventable blindness worldwide, in part through mass antibiotic treatment. DNA‐based nucleic acid amplification tests (NAATs) are currently used to evaluate the success of treatment programmes by measuring the prevalence of C trachomatis infection. Some believe that newer ribosomal RNA (rRNA)‐based tests may be much more sensitive since bacterial rRNA is present in amounts up to 10 000 times that of genomic DNA. Others believe that rRNA‐based tests are instead less sensitive but more specific, due to the presence of dead or subviable organisms that the test may not detect. This study compares an rRNA‐based test to a DNA‐based test for the detection of ocular C trachomatis infection in children living in trachoma‐endemic villages. Methods An rRNA‐based amplification test and DNA‐based polymerase chain reaction (PCR) were performed on swab specimens taken from the right upper tarsal conjunctiva of 56 children aged 0–10 years living in two villages in Amhara, Ethiopia. Results The rRNA‐based test detected ocular C trachomatis infection in 35 (63%) subjects compared with 22 (39%) detected by PCR (McNemar's test, p = 0.0002). The rRNA‐based test gave positive results for all subjects that were positive by PCR, and also detected infection in 13 (23%) additional subjects. Conclusion The rRNA‐based test appears to have significantly greater sensitivity than PCR for the detection of ocular chlamydial infection in children in trachoma‐endemic villages. Using the rRNA‐based test, we may be able to detect infection that was previously missed with PCR. Past studies using DNA‐based tests to assess prevalence of infectious trachoma following antibiotic treatment may have underestimated the true prevalence of infection. PMID:17050583

  10. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    Mauk, Michael G.; Changchun Liu; Jinzhao Song; Bau, Haim H

    2015-01-01

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (poly...

  11. Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

    Rodrigo Otávio Silveira Silva

    2014-07-01

    Full Text Available Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs and one nucleic acid amplification test (NAAT were evaluated against a cytotoxicity assay (CTA and toxigenic culture (TC. Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2% of 92 samples were positive according to the CTA, and 23 (25% were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

  12. Cost analysis of a nucleic acid amplification test in the diagnosis of pulmonary tuberculosis at an urban hospital with a high prevalence of TB/HIV.

    Max W Adelman

    Full Text Available INTRODUCTION: The Centers for Disease Control and Prevention has recommended using a nucleic acid amplification test (NAAT for diagnosing pulmonary tuberculosis (TB but there is a lack of data on NAAT cost-effectiveness. METHODS: We conducted a prospective cohort study that included all patients with an AFB smear-positive respiratory specimen at Grady Memorial Hospital in Atlanta, GA, USA between January 2002 and June 2008. We determined the sensitivity, specificity, and positive and negative predictive value of a commercially available and FDA-approved NAAT (amplified MTD, Gen-Probe compared to the gold standard of culture. A cost analysis was performed and included costs related to laboratory tests, hospital charges, anti-TB medications, and contact investigations. Average cost per patient was calculated under two conditions: (1 using a NAAT on all AFB smear-postive respiratory specimens and (2 not using a NAAT. One-way sensitivity analyses were conducted to determine sensitivity of cost difference to reasonable ranges of model inputs. RESULTS: During a 6 1/2 year study period, there were 1,009 patients with an AFB smear-positive respiratory specimen at our public urban hospital. We found the NAAT to be highly sensitive (99.6% and specific (99.1% on AFB smear-positive specimens compared to culture. Overall, the positive predictive value (PPV of an AFB smear-positive respiratory specimen for culture-confirmed TB was 27%. The PPV of an AFB smear-positive respiratory specimen for culture-confirmed TB was significantly higher for HIV-uninfected persons compared to those who were HIV-seropositive (152/271 [56%] vs. 85/445 [19%]; RR = 2.94, 95% CI 2.36-3.65, p<0.001. The cost savings of using the NAAT was $2,003 per AFB smear-positive case. CONCLUSIONS: Routine use of the NAAT on AFB smear-positive respiratory specimens was highly cost-saving in our setting at a U.S. urban public hospital with a high prevalence of TB and HIV because of the low

  13. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    Michael G. Mauk

    2015-10-01

    Full Text Available Microfluidic components and systems for rapid (<60 min, low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs are described. A microfluidic point-of-care (POC diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1 nucleic acids (NAs are extracted from relatively large (~mL volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane” to capture sample NAs in a flow-through, filtration mode; (2 NAs captured on the membrane are isothermally (~65 °C amplified; (3 amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4 paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  14. Participation of deoxyribonucleic acid polymerase alpha in amplification of ribosomal deoxyribonucleic acid in Xenopus laevis.

    Zimmermann, W.; Weissbach, A

    1981-01-01

    Aphidicolin, a known inhibitor of eucaryotic deoxyribonucleic acid (DNA) polymerase alpha, efficiently inhibited amplification of ribosomal DNA during oogenesis in Xenopus laevis. DNA polymerase alpha, but not DNA polymerase gamma, as isolated from ovaries, was sensitive to aphidicolin. DNA polymerase beta was not detectable in Xenopus ovary extracts. Therefore, DNA polymerase alpha plays a major role in ribosomal ribonucleic acid gene amplification.

  15. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.

    Lambert, Amy J; Martin, Denise A; Lanciotti, Robert S

    2003-01-01

    We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs. PMID:12517876

  16. Clostridium difficile Testing Algorithms Using Glutamate Dehydrogenase Antigen and C. difficile Toxin Enzyme Immunoassays with C. difficile Nucleic Acid Amplification Testing Increase Diagnostic Yield in a Tertiary Pediatric Population

    Ota, Kaede V.; McGowan, Karin L.

    2012-01-01

    We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined a...

  17. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

    Pascal Craw

    2015-09-01

    Full Text Available Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.

  18. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification.

    Craw, Pascal; Mackay, Ruth E; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S Tariq; Balachandran, Wamadeva

    2015-01-01

    Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings. PMID:26389913

  19. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

    Hummelshoj, Lone; Ryder, Lars P; Madsen, Hans O;

    2005-01-01

    both PCR and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking......Locked nucleic acid (LNA) is a modified DNA with increased binding affinityfor complementary DNA sequences. Our strategy was to use this property of LNA to inhibit undesired PCR amplification (e.g.,from contaminating genomic DNA) in a cDNA-based assay. By placing a short complementary LNA sequence...... activity. More than three DNA nucleotides reduced the LNA inhibition ability. The sequence specificity of the LNA was tested by investigating the number of LNA nucleotide mismatches permitted. The introduction of one mismatch maintained the inhibition of genomic amplification whereas two mismatches reduced...

  20. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics.

    Linnes, J C; Rodriguez, N M; Liu, L; Klapperich, C M

    2016-04-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications. PMID:26906904

  1. Clostridium difficile testing algorithms using glutamate dehydrogenase antigen and C. difficile toxin enzyme immunoassays with C. difficile nucleic acid amplification testing increase diagnostic yield in a tertiary pediatric population.

    Ota, Kaede V; McGowan, Karin L

    2012-04-01

    We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens. PMID:22259201

  2. Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

    Rodríguez-Lázaro, David; D'Agostino, Martin; Pla, Maria; Cook, Nigel

    2005-01-01

    An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nu...

  3. Nuclemeter: a reaction-diffusion based method for quantifying nucleic acids undergoing enzymatic amplification.

    Liu, Changchun; Sadik, Mohamed M; Mauk, Michael G; Edelstein, Paul H; Bushman, Frederic D; Gross, Robert; Bau, Haim H

    2014-01-01

    Real-time amplification and quantification of specific nucleic acid sequences plays a major role in medical and biotechnological applications. In the case of infectious diseases, such as HIV, quantification of the pathogen-load in patient specimens is critical to assess disease progression and effectiveness of drug therapy. Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings. This paper describes a simple, low-cost, reaction-diffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. The method was tested for HIV viral load monitoring and performed on par with conventional benchtop methods. The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free. PMID:25477046

  4. Isothermal amplification detection of nucleic acids by a double-nicked beacon.

    Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

    2016-03-01

    Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use. PMID:26706801

  5. Stomach acid test

    Gastric acid secretion test ... The test is done after you have not eaten for a while so fluid is all that remains in ... injected into your body. This is done to test the ability of the cells in the stomach ...

  6. Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories.

    Noordhoek, G T; van Embden, J D; Kolk, A H

    1996-01-01

    Nucleic acid amplification to detect Mycobacterium tuberculosis in clinical specimens is increasingly used as a laboratory tool for the diagnosis of tuberculosis. However, the specificity and sensitivity of these tests may be questioned, and no standardized reagents for quality control assessment are available. To estimate the performance of amplification tests for routine diagnosis, we initiated an interlaboratory study involving 30 laboratories in 18 countries. We prepared blinded panels of 20 sputum samples containing no, 100, or 1,000 mycobacterial cells. Each laboratory was asked to detect M. tuberculosis by their routine method of nucleic acid amplification. Only five laboratories correctly identified the presence or absence of mycobacterial DNA in all 20 samples. Seven laboratories detected mycobacterial DNA in all positive samples, and 13 laboratories correctly reported the absence of DNA in the negative samples. Lack of specificity was more of a problem than lack of sensitivity. Reliability was not found to be associated with the use of any particular method. Reliable detection of M. tuberculosis in clinical samples by nucleic acid amplification techniques is possible, but many laboratories do not use adequate quality controls. This study underlines the need for good laboratory practice and reference reagents to monitor the performance of the whole assay, including pretreatment of clinical samples. PMID:8880513

  7. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Olga A Gandelman

    Full Text Available BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a

  8. Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

    Zhan Sien

    2009-12-01

    Full Text Available Abstract Background Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV DNA levels, which can cause difficulty in the interpretation of viral load. A universal double-stranded DNA control or standard that can be used in all commercial HBV DNA nucleic acid amplification assay kits is urgently needed. By aligning all HBV genotypes (A-H, we found that the surface antigen gene and precore-core gene regions of HBV are the most conserved regions among the different HBV genotypes. We constructed a chimeric fragment by overlapping extension polymerase chain reaction and obtained a 1,349-bp HBVC+S fragment. We then packaged the fragment into lambda phages using a traditional lambda phage cloning procedure. Results The obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits. Conclusions The lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays.

  9. A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide

    Dames, Shale; Bromley, L. Kathryn; Herrmann, Mark; Elgort, Marc; Erali, Maria; Smith, Roger; Voelkerding, Karl V.

    2006-01-01

    A disposable 0.2-ml polymerase chain reaction (PCR) tube modified with an aluminum oxide membrane (AOM) has been developed for the extraction, amplification, and detection of nucleic acids. To assess the dynamic range of AOM tubes for real-time PCR, quantified herpes simplex virus (HSV) DNA was used to compare AOM tubes to standard PCR tubes. AOM PCR tubes used for amplification and detection of quantified HSV-1 displayed a crossing threshold (CT) shift 0.1 cycles greater than PCR tube contro...

  10. A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples.

    Rodriguez, Natalia M; Wong, Winnie S; Liu, Lena; Dewar, Rajan; Klapperich, Catherine M

    2016-02-21

    Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps into a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in less than 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings. PMID:26785636

  11. Reliability of nucleic acid amplification methods for detection of Chlamydia trachomatis in urine: results of the first international collaborative quality control study among 96 laboratories

    R.P.A.J. Verkooyen (Roel); G.T. Noordhoek; P.E. Klapper; J. Reid; J. Schirm; G.M. Cleator; M. Ieven; G. Hoddevik

    2003-01-01

    textabstractThe first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect Chlamydia trachomatis in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, includ

  12. Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends

    Zhang, Chunsun; Xing, Da

    2007-01-01

    The possibility of performing fast and small-volume nucleic acid amplification and analysis on a single chip has attracted great interest. Devices based on this idea, referred to as micro total analysis, microfluidic analysis, or simply ‘Lab on a chip’ systems, have witnessed steady advances over the last several years. Here, we summarize recent research on chip substrates, surface treatments, PCR reaction volume and speed, architecture, approaches to eliminating cross-contamination and contr...

  13. Isothermal cycling and cascade signal amplification strategy for ultrasensitive colorimetric detection of nucleic acids

    We have designed a novel isothermal cascade signal-amplification strategy for ultrasensitive colorimetric determination of nucleic acids. It is based on double-cycling amplification with formation of DNAzyme via a polymerase-induced strand-displacement reaction and nicking endonuclease-assisted recycling. The assay makes use of a hairpin DNA, a short primer, KF-polymerase, and nicking endonuclease. The presence of a target DNA triggers the strand-displacement and polymerization reaction with the formation of numerous DNAzyme molecules. Upon addition of H2O2 to the resulting mixture, the H2O2 reacts with 2,2′-azino-bis (3-ethylbenzothiozoline)-6-sulfonate to form a colored product in the aid of DNAzyme, which is quantified by photometry at 415 nm. Under optimal conditions, the assay allows target DNA to be determined at concentration as low as 0.6 aM. (author)

  14. Onium salts as radiation-sensitive acid generators for resists with chemical amplification (review)

    The necessity of mastering alternative lithographic processes for development and production of 64 and 256 Mbit dynamic random-access memory units has been substantiated. It is shown that conventional positive photoresists based on diazonaphthoquinone and novalac resins do not meet the requirements of modern microlithography. The concept of chemical amplification offered a means for developing adequate topological structures with sizes of resist components of 0.35 μm or less. Onium salts are universal and efficient acid generators for resists with chemical amplification. Studies in the field of photo- and radiochemistry of onium salts have been summarized and correlated. It has been shown that the quantum yield and distribution of photolysis products are governed to a major extent by geminal and bulk recombination. Specific features of photolysis and radiolysis of onium salts in a polymer matrix are considered

  15. Nuclemeter: A Reaction-Diffusion Column for Quantifying Nucleic Acids Undergoing Enzymatic Amplification

    Bau, Haim; Liu, Changchun; Killawala, Chitvan; Sadik, Mohamed; Mauk, Michael

    2014-11-01

    Real-time amplification and quantification of specific nucleic acid sequences plays a major role in many medical and biotechnological applications. In the case of infectious diseases, quantification of the pathogen-load in patient specimens is critical to assessing disease progression, effectiveness of drug therapy, and emergence of drug-resistance. Typically, nucleic acid quantification requires sophisticated and expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low resource settings. We describe a simple, low-cost, reactiondiffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. We model the process with the Fisher Kolmogoroff Petrovskii Piscounoff (FKPP) Equation and compare theoretical predictions with experimental observations. The proposed method is suitable for nucleic acid quantification at the point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free. C.L. was supported by NIH/NIAID K25AI099160; M.S. was supported by the Pennsylvania Ben Franklin Technology Development Authority; C.K. and H.B. were funded, in part, by NIH/NIAID 1R41AI104418-01A1.

  16. Effect of basic additives on sensitivity and diffusion of acid in chemical amplification resists

    Asakawa, Koji; Ushirogouchi, Tohru; Nakase, Makoto

    1995-06-01

    The effect of amine additives in chemical amplification resists is discussed. Phenolic amines such as 4-aminophenol and 2-(4-aminophenyl)-2-(4-hydroxyphenyl) propane were investigated as model compounds from the viewpoint of sensitivity, diffusion and resolution. Equal molar amounts of acid and amine deactivated at the very beginning of post-exposure bake, and could not participate in decomposing the inhibitor as a catalyst. Only the acid which survived from the deactivation diffuses in the resist, decomposing the inhibitors from the middle to late stage of PEB. The basic additives reduce the diffusion range of the acid, especially for long-range diffusion, resulting in higher contrast at the interfaces between the exposed and unexposed areas. In addition, the amine concentration required is found to be less than the concentration which causes the resist sensitivity to start decreasing.

  17. Test of the depression distress amplification model in young adults with elevated risk of current suicidality.

    Capron, Daniel W; Lamis, Dorian A; Schmidt, Norman B

    2014-11-30

    Suicide is a leading cause of death among young adults and the rate of suicide has been increasing for decades. A depression distress amplification model posits that young adults with comorbid depression and anxiety have elevated suicide rates due to the intensification of their depressive symptoms by anxiety sensitivity cognitive concerns. The current study tested the effects of anxiety sensitivity subfactors as well as the depression distress amplification model in a very large sample of college students with elevated suicide risk. Participants were 721 college students who were at elevated risk of suicidality (scored>0 on the Beck Scale for Suicide Ideation). Consistent with prior work, anxiety sensitivity cognitive concerns, but not physical or social concerns, were associated with suicidal ideation. Consistent with the depression distress amplification model, in individuals high in depression, anxiety sensitivity cognitive concerns predicted elevated suicidal ideation but not among those with low depression. The results of this study corroborate the role of anxiety sensitivity cognitive concerns and the depression distress amplification model in suicidal ideation among a large potentially high-risk group of college students. The depression distress amplification model suggests a specific mechanism, anxiety sensitivity cognitive concerns, that may be responsible for increased suicide rates among those with comorbid anxiety and depression. PMID:25063018

  18. Nucleic acid amplification technology screening for hepatitis C virus and human immunodeficiency virus for blood donations

    To investigate the performance of the commercial Roche COBAS AmpliScreen assay, and demonstrate whether the COBAS AmpliScreen human immunodeficiency virus-1 (HIV-1) test, v1.5, and COBAS AmpliScreen hepatitis C virus (HCV) v 2.0 for screening for HIV-1 and HCV RNA in the donated blood units from which plasma mini pools were collected, by nucleic acid amplification technology (NAT), could detect the positive pools and reduce the risk of transmission of infections for those routinely tested by serological assays. The study was performed on 3288 plasma samples collected from blood donors in a period of 13 months, from August 2004 to August 2005, at Al-Hada Armed Forces Hospital, Molecular Pathology Laboratory, Taif, Kingdom of Saudi Arabia. The samples were tested by the reverse transcriptase polymerase chain reaction (RT-PCR) after RNA extraction (this represents the major method in NAT assays), in parallel with the routine serological testing to detect qualitatively for HIV-1 and HCV. The NAT assays that include an automated COBAS AmpliPrep system for RNA extraction and COBAS Amplicor Analyzer using AmpliScreen kits for RT-PCR assays, and the routine serological screening assays for the detection of the HIV-1 and HCV RNA in the plasma samples from the blood donors have shown to be a reliable combination that would meet our requirements. The collected data further confirms the results from the serological assays and enables us to decrease the residual risk of transmission to a minimum with the finding of no seronegative window period donation. The results demonstrate that out of 3288 samples, the percentages of RT-PCR (NAT) negative blood donations that were also confirmed as seronegative were 99% for HCV, and 99.1% for HIV-1. The modified combined systems (automated COBAS AmpliPrep system for RNA extraction and COBAS Amplicor Analyzer using AmpliScreen kits for RT-PCR assays) for NAT screening assays has allowed the release of all blood donations supplied in the

  19. System for portable nucleic acid testing in low resource settings

    Lu, Hsiang-Wei; Roskos, Kristina; Hickerson, Anna I.; Carey, Thomas; Niemz, Angelika

    2013-03-01

    Our overall goal is to enable timely diagnosis of infectious diseases through nucleic acid testing at the point-of-care and in low resource settings, via a compact system that integrates nucleic acid sample preparation, isothermal DNA amplification, and nucleic acid lateral flow (NALF) detection. We herein present an interim milestone, the design of the amplification and detection subsystem, and the characterization of thermal and fluidic control and assay execution within this system. Using an earlier prototype of the amplification and detection unit, comprised of a disposable cartridge containing flexible pouches, passive valves, and electrolysis-driven pumps, in conjunction with a small heater, we have demonstrated successful execution of an established and clinically validated isothermal loop-mediated amplification (LAMP) reaction targeting Mycobacterium tuberculosis (M.tb) DNA, coupled to NALF detection. The refined design presented herein incorporates miniaturized and integrated electrolytic pumps, novel passive valves, overall design changes to facilitate integration with an upstream sample preparation unit, and a refined instrument design that automates pumping, heating, and timing. Nucleic acid amplification occurs in a two-layer pouch that facilitates fluid handling and appropriate thermal control. The disposable cartridge is manufactured using low-cost and scalable techniques and forms a closed system to prevent workplace contamination by amplicons. In a parallel effort, we are developing a sample preparation unit based on similar design principles, which performs mechanical lysis of mycobacteria and DNA extraction from liquefied and disinfected sputum. Our next step is to combine sample preparation, amplification, and detection in a final integrated cartridge and device, to enable fully automated sample-in to answer-out diagnosis of active tuberculosis in primary care facilities of low-resource and high-burden countries.

  20. Reliability of Nucleic Acid Amplification Methods for Detection of Chlamydia trachomatis in Urine: Results of the First International Collaborative Quality Control Study among 96 Laboratories

    Verkooyen, Roel; Noordhoek, G T; Klapper, P.E.; Reid, J.; Schirm, J.; Cleator, G. M.; Ieven, M.; Hoddevik, G.

    2003-01-01

    textabstractThe first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect Chlamydia trachomatis in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, including three negative, two strongly positive, and five weakly positive samples. Ninety-six laboratories in 22 countries participated with a total of 102 data sets. Of 204 strongly positive samples 199 (97.5%) were corr...

  1. Clinical utility of a nested nucleic acid amplification format in comparison to viral culture for the diagnosis of mucosal herpes simplex infection in a genitourinary medicine setting

    Wyatt Dorothy E; McCaughey Conall; O'Neill Hugh J; Coyle Peter V; McBride Michael O

    2001-01-01

    Abstract Background Nested nucleic acid amplification tests are often thought too sensitive or prone to generatingfalse positive results for routine use. The current study investigated the specificity and clinicalutility of a routine multiplex nested assay for mucosal herpetic infections. Methods Ninety patients, categorised into those clinically diagnosed to (a) have and (b) not haveherpetic infection, were enrolled. Swabs from oral and ano-genital sites were assayed by thenested assay and c...

  2. Statistical hypothesis testing by weak-value amplification: Proposal and evaluation

    Susa, Yuki; Tanaka, Saki

    2015-01-01

    We study the detection capability of the weak-value amplification on the basis of the statistical hypothesis testing. We propose a reasonable testing method in the physical and statistical senses to find that the weak measurement with the large weak value has the advantage to increase the detection power and to reduce the possibility of missing the presence of interaction. We enhance the physical understanding of the weak value and mathematically establish the significance of the weak-value a...

  3. Citric acid urine test

    ... usually done while you are on a normal diet. Ask your provider for more information. ... acidosis and a tendency to form calcium kidney stones. The ... acid levels: A high carbohydrate diet Estrogen therapy Vitamin D

  4. Improved sensitivity of nucleic acid amplification for rapid diagnosis of tuberculous meningitis

    Johansen, Isik Somuncu; Lundgren, Bettina; Tabak, Fehmi; Petrini, Björn; Hosoglu, Salih; Saltoglu, Nese; Thomsen, Vibeke Østergaard

    2004-01-01

    Early diagnosis of tuberculous meningitis (TBM) is essential for a positive outcome; but present microbiological diagnostic techniques are insensitive, slow, or laborious. We evaluated the standard BDProbeTec ET strand displacement amplification method (the standard ProbeTec method) for the...... diagnosis was attained by culture. Thirteen specimens from 12 patients were culture positive for M. tuberculosis complex organisms; three specimens (23%) were microscopy positive for acid-fast bacilli. Among the culture-positive specimens, the standard ProbeTec method was positive for 8 (61.5%) and the...... modified assay was positive for 10 (76.9%). The overall specificity by both procedures was 98.8% compared to the results of culture. After discrepancy analysis, conducted by reviewing the patients' previous laboratory data, the specificity increased to 100%. If the cutoff value for respiratory specimens...

  5. Lactic acid test

    ... test is most often done to diagnose lactic acidosis . Normal Results 4.5 to 19.8 mg/ ... PA: Elsevier Saunders; 2016:chap 118. Read More Acidosis Aerobic Anaerobic Heart failure - overview Hepatic Metabolism Muscle ...

  6. Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples ▿ †

    Hata, Akihiko; Katayama, Hiroyuki; Kitajima, Masaaki; Visvanathan, Chettiyappan; Nol, Chea; Furumai, Hiroaki

    2011-01-01

    Inhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplifie...

  7. 4-(Dimethylamino)butyric acid labeling for electrochemiluminescence detection of biological substances by increasing sensitivity with gold nanoparticle amplification.

    Yin, Xue-Bo; Qi, Bin; Sun, Xuping; Yang, Xiurong; Wang, Erkang

    2005-06-01

    4-(Dimethylamino)butyric acid (DMBA) labeling combined with gold nanoparticle amplification for electrochemiluminescence (ECL) determination of a biological substance (bovine serum albumin (BSA) and immunoglobulin G (IgG) as models) was presented. After DMBA, an analogue of tripropylamine, was tagged on the (anti)analytes, an ECL signal related to the content of the analytes was generated when the analyte tagged with DMBA was in contact with tris(2,2'-bipyridine)ruthenium (Ru(bpy)(3)2+) solution and a potential was applied. To improve the adsorption capacity, a gold nanoparticle layer was first combined into the surface of the 2-mm-diameter gold electrode. For the determination of BSA, avidin was covalently conjugated to a self-assembled monolayer of 3-mercaptopropanoic acid on the gold nanoparticle layer. Biotinylated BSA-DMBA was then immobilized on the gold nanoparticle layer of the gold electrode via the avidin-biotin reaction. IgG was tested via a typical sandwich-type immobilization method. ECL signals were generated from the electrodes immobilized with BSA or IgG by immersing them in a 1 mmol L-1 Ru(bpy)(3)2+ solution and scanning from 0.5 to 1.3 V versus Ag/AgCl. With gold nanoparticle amplification, the ECL peak intensity was proportional to the concentration over the range 1-80 and 5-100 microg/mL for BSA and IgG consuming 50 microL of sample, respectively. A 10- and 6-fold sensitivity enhancement was obtained for BSA and IgG over their direct immobilization on an electrode using DMBA labeling. The relative standard deviations of five replicate determinations of 10 microg/mL BSA and 20 microg/mL IgG were 8.4 and 10.2%, respectively. High biocompatibility and low cost were the main advantages of the present DMBA labeling technique over the traditional Ru(bpy)(3)2+ labeling. PMID:15924384

  8. Prospective evaluation of the Alere i Influenza A&B nucleic acid amplification versus Xpert Flu/RSV.

    Nguyen Van, J C; Caméléna, F; Dahoun, M; Pilmis, B; Mizrahi, A; Lourtet, J; Behillil, S; Enouf, V; Le Monnier, A

    2016-05-01

    The rapid and accurate detection of influenza virus in respiratory specimens is required for optimal management of patients with acute respiratory infections. Because of the variability of the symptoms and the numerous other causes of influenza-like illness, the diagnosis of influenza cannot be made on the basis of clinical criteria alone. Thus, rapid influenza diagnostic tests have been developed such as the Alere i Influenza A&B isothermal nucleic acid assay. We prospectively evaluated the performance of the Alere i Influenza A&B assay in comparison with our routine Xpert Flu/RSV assay. Positive samples were subtyped according to the protocol from the National Influenza Center (Paris, France). A total of 96 respiratory nasal swab samples were analyzed: with both methods, 38 were positive and 56 were negative. Samples were prospectively collected from January 20 to April 8, 2015, from patient (86 adult and 10 pediatric patients) presenting with an influenza-like illness through the French influenza season. In comparison with the Xpert Flu/RSV assay, the overall sensitivity and specificity of the Alere i Influenza A&B assay were 95% and 100%, respectively. Our results indicate that the Alere i Influenza A&B assay has a good overall analytical performance and a high degree of concordance with the PCR-based Xpert Flu/RSV assay. The Alere i Influenza A&B isothermal nucleic acid amplification test is a powerful tool for influenza detection due to its high sensitivity and specificity as well as its ability to generate results within 15min. PMID:26899154

  9. Molecular Investigation of Lymph Nodes in Colon Cancer Patients Using One-Step Nucleic Acid Amplification (OSNA)

    Güller, Ulrich; Zettl, Andreas; Worni, Mathias; Langer, Igor; Cabalzar-Wondberg, Daniela; Viehl, Carsten T; Demartines, Nicolas; Zuber, Markus

    2012-01-01

    BACKGROUND A new diagnostic system, called one-step nucleic acid amplification (OSNA), has recently been designed to detect cytokeratin 19 mRNA as a surrogate for lymph node metastases. The objective of this prospective investigation was to compare the performance of OSNA with both standard hematoxylin and eosin (H&E) analysis and intensive histopathology in the detection of colon cancer lymph node metastases. METHODS In total, 313 lymph nodes from 22 consecutive patients with stage I, II, an...

  10. Real-Time Nucleic Acid Sequence-Based Amplification Using Molecular Beacons for Detection of Enterovirus RNA in Clinical Specimens

    Landry, Marie L.; Garner, Robin; Ferguson, David

    2005-01-01

    Real-time nucleic acid sequence-based amplification (NASBA) using molecular beacon technology (NASBA-beacon) was compared to standard NASBA with postamplification hybridization using electrochemiluminescently labeled probes (NASBA-ECL) for detection of enteroviruses (EV) in 133 cerebrospinal fluid and 27 stool samples. NASBA-ECL and NASBA-beacon were similar in sensitivity, detecting 55 (100%) and 52 (94.5%) EV-positive samples, respectively. There were no false positives. Both NASBA assays w...

  11. HER-2/neu amplification testing in breast cancer by Multiplex Ligation-dependent Probe Amplification: influence of manual- and laser microdissection

    Accurate assessment of HER-2/neu status is crucial for proper prognostic information and to offer direct appropriate treatment for breast cancer patients. Next to immunohistochemistry (IHC) to evaluate HER2 protein overexpression, a second line gene amplification test is generally deemed necessary for cases with equivocal protein expression. Recently, a new PCR based test, called Multiplex Ligation-dependent Probe Amplification (MLPA), was introduced as a simple and quick method to assess HER-2/neu gene amplification status in invasive breast cancer. MLPA was previously shown to correlate well with IHC and in situ hybridization (ISH), but a low tumor percentage in the tissue tested could negatively affect the accuracy of MLPA results. To examine this, MLPA was repeated in 42 patients after serial H&E section guided manual dissection with a scalpel and after laser microdissection of the tumor. Both dissection techniques led to higher HER2 gene copy number ratios and thereby made MLPA more quantitative. Concordance between MLPA and ISH improved from 61% to 84% after manual microdissection and to 90% after laser microdissection. Manual and laser microdissection similarly increase the dynamic range of MLPA copy number ratios which is a technical advantage. As clinically a dichotomization between normal and amplified suffices and MLPA is relatively unsensitive to tumor content, microdissection before MLPA may not be routinely necessary but may be advisable in case of very low tumor content (≤30%), when MLPA results are equivocal, or when extensive ductal carcinoma in situ is present. Since differences between manual and laser microdissection were small, less time consuming manual microdissection appears to be sufficient

  12. Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification

    Yi Wang; Yan Wang; Ai-Jing Ma; Dong-Xun Li; Li-Juan Luo; Dong-Xin Liu; Dong Jin; Kai Liu; Chang-Yun Ye

    2015-01-01

    We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61–65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primer...

  13. A simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings.

    Paul LaBarre

    Full Text Available BACKGROUND: Molecular assays targeted to nucleic acid (NA markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR instrumentation (another is sample preparation. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. CONCLUSIONS/SIGNIFICANCE: We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes.

  14. Comparison of nucleic acid hybridization and nucleic acid amplification using conserved sequences from the 5' noncoding region for detection of bovine viral diarrhea virus.

    Ridpath, J F; Bolin, S R; Katz, J

    1993-01-01

    Primers and probes derived from conserved sequences located in the 5' noncoding region of pestiviruses were evaluated for detection of bovine viral diarrhea virus. With these reagents, hybridization and polymerase chain reaction tests detected 62 of 90 and 90 of 90 bovine viral diarrhea virus isolates, respectively. A quick lysis method for preparing RNA for use in polymerase chain reaction amplification also was evaluated.

  15. Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

    Xu, Changping; Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Cao, Zengguo; Li, Ling; Wang, Jianzhong; Yan, Feihu; Wang, Lina; Chi, Hang; Gai, Weiwei; Wang, Chong; Zhao, Yongkun; Feng, Yan; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions. PMID:26831931

  16. Physical description of statistical hypothesis testing for a weak-value amplification experiment using a birefringent crystal

    Susa, Yuki

    2015-01-01

    We investigate the weak measurement experiment demonstrated by Ritchie et al. [N. W. M. Ritchie, J. G. Story, and R. G. Hulet, Phys. Rev. Lett. 66, 1107 (1991)] from the viewpoint of the statistical hypothesis testing for the weak-value amplification proposed by Susa and Tanaka [Y. Susa and S. Tanaka, Phys. Rev. A 92, 012112 (2015)]. We conclude that the weak-value amplification is a better method to determine whether the crystal used in the experiment is birefringent than the measurement wit...

  17. Novel Real-Time Simultaneous Amplification and Testing Method To Accurately and Rapidly Detect Mycobacterium tuberculosis Complex

    Cui, Zhenling; Wang, Yongzhong; Fang, Liang; Zheng, Ruijuan; Huang, Xiaochen; Liu, Xiaoqin; Zhang, Gang; Rui, Dongmei; Ju, Jinliang; Hu, Zhongyi

    2012-01-01

    The aim of this study was to establish and evaluate a simultaneous amplification and testing method for detection of the Mycobacterium tuberculosis complex (SAT-TB assay) in clinical specimens by using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB assay, a 170-bp M. tuberculosis 16S rRNA fragment is reverse transcribed to DNA by use of Moloney murine leukemia virus (M-MLV) reverse transcriptase, using specific primers incorporating the T7 promoter sequence, ...

  18. Whole genome amplification induced bias in the detection of KRAS-mutated cell populations during colorectal carcinoma tissue testing.

    Stranska, Jana; Jancik, Sylwia; Slavkovsky, Rastislav; Holinkova, Veronika; Rabcanova, Miroslava; Vojta, Petr; Hajduch, Marian; Drabek, Jiri

    2015-03-01

    Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA. We analyzed the origins of discrepancies in KRAS (Kirsten rat sarcoma viral oncogene homolog gene) mutation detection in six of ten samples amplified using the GenomePlex® Tissue Whole Genome Amplification kit 5 (WGA5; Sigma-Aldrich, St. Louis, MO, USA) and KRAS StripAssay® (KRAS SA; ViennaLab Diagnostics, Vienna, Austria). We undertook reextraction, reamplification, retyping, authentication, reanalysis, and reinterpretation to determine whether the discrepancies originated during the preanalytical, analytical, and/or interpretative phase of genotyping. We conclude that a combination of glass slide/sample heterogeneity and biased amplification due to stochastic effects in the early phases of whole genome amplification (WGA) may have adversely affected the results obtained. Our findings are relevant for both forensic genetics testing and massively parallel sequencing using preamplification. PMID:25655305

  19. Improved sensitivity of nucleic acid amplification for rapid diagnosis of tuberculous meningitis

    Johansen, Isik Somuncu; Lundgren, Bettina; Tabak, Fehmi; Petrini, Björn; Hosoglu, Salih; Saltoglu, Nese; Thomsen, Vibeke Østergaard

    2004-01-01

    Early diagnosis of tuberculous meningitis (TBM) is essential for a positive outcome; but present microbiological diagnostic techniques are insensitive, slow, or laborious. We evaluated the standard BDProbeTec ET strand displacement amplification method (the standard ProbeTec method) for the detec...

  20. Two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites.

    Liu, Qing; Nam, Jeonghun; Kim, Sangho; Lim, Chwee Teck; Park, Mi Kyoung; Shin, Yong

    2016-08-15

    Rapid, early, and accurate diagnosis of malaria is essential for effective disease management and surveillance, and can reduce morbidity and mortality associated with the disease. Although significant advances have been achieved for the diagnosis of malaria, these technologies are still far from ideal, being time consuming, complex and poorly sensitive as well as requiring separate assays for sample processing and detection. Therefore, the development of a fast and sensitive method that can integrate sample processing with detection of malarial infection is desirable. Here, we report a two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites. It combines the Dimethyl adipimidate (DMA)/Thin film Sample processing (DTS) technique as a first stage and the Mach-Zehnder Interferometer-Isothermal solid-phase DNA Amplification (MZI-IDA) sensing technique as a second stage. The system can extract DNA from malarial parasites using DTS technique in a closed system, not only reducing sample loss and contamination, but also facilitating the multiplexed malarial DNA detection using the fast and accurate MZI-IDA technique. Here, we demonstrated that this system can deliver results within 60min (including sample processing, amplification and detection) with high sensitivity (malaria in low-resource settings. PMID:27031184

  1. Rapid and sensitive loop-mediated isothermal amplification test for Clostridium difficile detection challenges cytotoxin B cell test and culture as gold standard.

    Norén, Torbjörn; Alriksson, Ingegärd; Andersson, Josefin; Akerlund, Thomas; Unemo, Magnus

    2011-02-01

    Compared to the composite gold standard cytotoxin B assay and toxigenic culture, the loop-mediated isothermal amplification (LAMP) test for Clostridium difficile had a sensitivity and specificity of 98%, positive predictive value of 92%, and negative predictive value of >99%. A one-hour turnaround time for the LAMP test provides rapid diagnosis and cost savings. PMID:21106782

  2. Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for Clostridium difficile Detection Challenges Cytotoxin B Cell Test and Culture as Gold Standard▿

    Norén, Torbjörn; Alriksson, Ingegärd; Andersson, Josefin; Åkerlund, Thomas; Unemo, Magnus

    2011-01-01

    Compared to the composite gold standard cytotoxin B assay and toxigenic culture, the loop-mediated isothermal amplification (LAMP) test for Clostridium difficile had a sensitivity and specificity of 98%, positive predictive value of 92%, and negative predictive value of >99%. A one-hour turnaround time for the LAMP test provides rapid diagnosis and cost savings.

  3. Label-free electrochemical nucleic acid biosensing by tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme amplification.

    Liu, Shufeng; Gong, Hongwei; Wang, Yanqun; Wang, Li

    2016-03-15

    Owing to the intrinsic importance of nucleic acid as bio-targets, the achievement of its simple and sensitive detection with high confidence is very essential for biological studies and diagnostic purposes. Herein, a label-free, isothermal, and ultrasensitive electrochemical detection of target DNA was developed by using a tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme releasing amplification strategy. Upon sensing of the nucleic acid analyte for the assembled hairpin-like probe DNA on the electrode, the DNA polymerase guided the target recycling and simultaneously triggered the lambda exonuclease cleavage, accompanied by the cascade recycling of the released new complementary strand and the amplified liberation of the G-rich sequence of the HRP-mimicking DNAzyme. The electrocatalytic reduction of H2O2 by the generated hemin/G-quadruplex DNAzyme was used for the signal readout and further amplification toward target response. Such tandem functional operation by DNA polymerase, lambda exonuclease and DNAzyme endows the developed biosensor with a high sensitivity and also a high confidence. A low detection limit of 5 fM with an excellent selectivity toward target DNA could be achieved. It also exhibits the distinct advantages of simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus may offer a promising avenue for the applications in disease diagnosis and clinical biomedicine. PMID:26513289

  4. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  5. Competitive amplification of differentially melting amplicons (CADMA improves KRAS hotspot mutation testing in colorectal cancer

    Kristensen Lasse

    2012-11-01

    Full Text Available Abstract Background Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR. Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. Methods We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA. The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. Results The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. Conclusions The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

  6. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    Anja Gulliksen

    2012-01-01

    Full Text Available The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n=28 from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

  7. Chip-based device for parallel sorting, amplification, detection, and identification of nucleic acid subsequences

    Beer, Neil Reginald; Colston, Jr, Billy W.

    2016-08-09

    An apparatus for chip-based sorting, amplification, detection, and identification of a sample having a planar substrate. The planar substrate is divided into cells. The cells are arranged on the planar substrate in rows and columns. Electrodes are located in the cells. A micro-reactor maker produces micro-reactors containing the sample. The micro-reactor maker is positioned to deliver the micro-reactors to the planar substrate. A microprocessor is connected to the electrodes for manipulating the micro-reactors on the planar substrate. A detector is positioned to interrogate the sample contained in the micro-reactors.

  8. Acid Tests and Basic Fun.

    McBride, John W.

    1995-01-01

    Explores acids and bases using different indicators, such as turmeric, purple grape juice, and lichens. Because some of these indicators are not as sensitive as cabbage juice or litmus paper, determining to which acids and bases each indicator is sensitive presents an enjoyable, problem-solving challenge for students. Presents directions for…

  9. Detection of Staphylococcus epidermidis by a Quartz Crystal Microbalance Nucleic Acid Biosensor Array Using Au Nanoparticle Signal Amplification

    Weiling Fu

    2008-10-01

    Full Text Available Staphylococcus epidermidis is a critical pathogen of nosocomial blood infections, resulting in significant morbidity and mortality. A piezoelectric quartz crystal microbalance (QCM nucleic acid biosensor array using Au nanoparticle signal amplification was developed to rapidly detect S. epidermidis in clinical samples. The synthesized thiolated probes specific targeting S. epidermidis 16S rRNA gene were immobilized on the surface of QCM nucleic acid biosensor arrays. Hybridization was induced by exposing the immobilized probes to the PCR amplified fragments of S. epidermidis, resulting in a mass change and a consequent frequency shift of the QCM biosensor. To further enhance frequency shift results from above described hybridizations, streptavidin coated Au nanoparticles were conjugated to the PCR amplified fragments. The results showed that the lowest detection limit of current QCM system was 1.3×103 CFU/mL. A linear correlation was found when the concentration of S. epidermidis varied from 1.3×103 to 1.3×107 CFU/mL. In addition, 55 clinical samples were detected with both current QCM biosensor system and conventional clinical microbiological method, and the sensitivity and specificity of current QCM biosensor system were 97.14% and 100%, respectively. In conclusion, the current QCM system is a rapid, low-cost and sensitive method that can be used to identify infection of S. epidermidis in clinical samples.

  10. An ultrasensitive electrochemical immunosensor platform with double signal amplification for indole-3-acetic acid determinations in plant seeds.

    Yin, Huanshun; Xu, Zhenning; Zhou, Yunlei; Wang, Mo; Ai, Shiyun

    2013-03-21

    A label-free electrochemical immunosensor for ultra-sensitive detection of indole-3-acetic acid (IAA), a very important phytohormone, has been developed in this work. The detection strategy was mainly based on 4-aminophenylboronic acid, magnetic nanoparticles functionalized with horseradish peroxidase-conjugated goat anti-rat immunoglobulin G (HRP-IgG-Fe(3)O(4)) and rat monoclonal antibody against IAA-modified gold nanoparticles (anti-IAA-AuNPs). HRP-IgG-AuNPs was covalently assembled on the electrode surface through the specific chemical reaction between boronic acid and the vicinal diol in HRP-IgG. Then, anti-IAA-AuNPs was further assembled on the electrode via the interaction between IgG and antibody. Through the dual amplification of HRP-IgG-Fe(3)O(4) and anti-IAA-AuNPs, the trapping capacity of the immunosensor for IAA was significantly enhanced based on the promotion of the immunoreaction between antibody and antigen, which resulted in a large decrease of the electrochemical response of the redox probe, Fe(CN)(6)(3-), and an increase in sensitivity. The developed electrochemical immunosensor exhibited a wide linear range from 0.02 to 500 ng mL(-1) with a low detection limit of 0.018 ng mL(-1) (S/N = 3). Moreover, the proposed immunosensor showed acceptable selectivity, reproducibility, accuracy and stability. The IAA extracted from various seeds was successfully detected using the developed immunosensor. This assay method might provide an alternative strategy for the detection of various phytohormones. PMID:23377501

  11. 21 CFR 862.1450 - Lactic acid test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactic acid test system. 862.1450 Section 862.1450....1450 Lactic acid test system. (a) Identification. A lactic acid test system is a device intended to measure lactic acid in whole blood and plasma. Lactic acid measurements that evaluate the acid-base...

  12. Multicenter evaluation of the Quidel Lyra Direct C. difficile nucleic acid amplification assay.

    Beck, Eric T; Buchan, Blake W; Riebe, Katherine M; Alkins, Brenda R; Pancholi, Preeti; Granato, Paul A; Ledeboer, Nathan A

    2014-06-01

    Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods. PMID:24671790

  13. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification

    Silahtaroglu, Asli; Nolting, Dorrit; Andersen, Lars Dyrskjøt; Berezikov, Eugene; Møller, Morten; Tommerup, Niels; Kauppinen, Markus Sakari

    2007-01-01

    RNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have...... protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution....

  14. Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure

    Wharam, Susan D.; Marsh, Peter; Lloyd, John S.; Ray, Trevor D.; Mock, Graham A.; Assenberg, René; McPhee, Julie E.; Brown, Philip; Weston, Anthony; Cardy, Donald L. N.

    2001-01-01

    The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This ...

  15. A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections

    Feeney Susan A; Mitchell Suzanne J; Mitchell Frederick; De Ornellas Dennis; McCaughey Conall; O'Neill Hugh J; Ong Grace M; Coyle Peter V; Wyatt Dorothy E; Forde Marian; Stockton Joanne

    2004-01-01

    Abstract Background Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 c...

  16. Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season 2014/15.

    Huzly, Daniela; Korn, Klaus; Bierbaum, Sibylle; Eberle, Björn; Falcone, Valeria; Knöll, Antje; Steininger, Philipp; Panning, Marcus

    2016-09-01

    The influenza season 2014/15 was dominated by drift variants of influenza A(H3N2), which resulted in a reduced vaccine effectiveness. It was not clear if the performance of commercial nucleic-acid-based amplification (NAT) assays for the detection of influenza was affected. The purpose of this study was to perform a real-life evaluation of two commercial NAT assays. During January-April 2015, we tested a total of 665 samples from patients with influenza-like illness using the Fast Track Diagnostics Respiratory pathogens 21, a commercial multiplex kit, (cohorts 1 and 2, n = 563 patients) and the Xpert Flu/RSV XC assay (cohort 3, n = 102 patients), a single-use cartridge system. An in-house influenza real-time RT-PCR (cohort 1) and the RealStar Influenza RT-PCR 1.0 Kit (cohort 2 and 3) served as reference tests. Compared to the reference assay, an overall agreement of 95.9 % (cohort 1), 95 % (cohort 2), and 98 % (cohort 3) was achieved. A total of 24 false-negative results were observed using the Fast Track Diagnostics Respiratory pathogens 21 kit. No false-negative results occurred using the Xpert Flu/RSV XC assay. The Fast Track Diagnostics Respiratory pathogens 21 kit and the Xpert Flu/RSV XC assay had sensitivities of 90.7 % and 100 % and specificities of 100 % and 94.1 %, respectively, compared to the RealStar 1.0 kit. Upon modification of the Fast Track Diagnostics Respiratory pathogens 21 kit, the sensitivity increased to 97.3 %. Influenza virus strains circulating during the 2014/15 season reduced the detection sensitivity of a commercial NAT assay, and continuous monitoring of test performance is therefore necessary. PMID:27316440

  17. Substituted Indoleacetic Acids Tested in Tissue Cultures

    Engvild, Kjeld Christensen

    1978-01-01

    Monochloro substituted IAA inhibited shoot induction in tobacco tissue cultures about as much as IAA. Dichloro substituted IAA inhibited shoot formation less. Other substituted IAA except 5-fluoro- and 5-bromoindole-3-acetic acid were less active than IAA. Callus growth was quite variable and not...... and not correlated with auxin strength measured in the Avena coleoptile test....

  18. Ultrasensitive electrochemical detection of nucleic acid by coupling an autonomous cascade target replication and enzyme/gold nanoparticle-based post-amplification.

    Liu, Shufeng; Wei, Wenji; Wang, Yanqun; Fang, Li; Wang, Li; Li, Feng

    2016-06-15

    Owing to the intrinsic importance of nucleic acid as bio-targets, the development of isothermal and ultrasensitive electrochemical DNA biosensor is very essential for biological studies and medical diagnostics. Herein, the autonomous cascade DNA replication strategy was effectively married with the enzyme/gold nanoparticle-based post-amplification strategy to promote the detection performance toward target DNA. A hairpin DNA probe (HP) is designed that consists of an overhang at 3'-end as the recognition unit for target DNA, a recognition site for nicking endonuclease, and an alkane spacer to terminate polymerization reaction. The autonomous DNA replication-scission-displacement reaction operated by the nicking endonuclease/KF polymerase induced the autocatalytic opening of HP, which was then specifically bound by the enzyme/gold nanoparticles for further dual-signal amplification toward target-related sensing events. A low detection limit of 0.065fM with an excellent selectivity toward target DNA could be achieved. The proposed biosensor could be also easily regenerated for target detection. The developed biosensor creates an opportunity for the effective coupling of the target replication with post-amplification strategies and thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. PMID:26849348

  19. Evaluation of two DNA amplification PCR tests for the diagnosis of Clostridium difficile infection

    Rita Caldarelli

    2010-09-01

    Full Text Available Introduction. Clostridium Difficile (CD usually is present in the gut of healthy subjects without giving any disease. As a consequence of various stress, including antibiotic therapy, CD can replicate and produce A and B toxins that induce diarrhoea.The finding of A and B toxins is a landmark for diagnosis of CD infection. Methods. 60 stool samples have been tested for CD presence. All the samples have been tested for the glutamate dehydrogenase (GDH presence.The GDH positive samples have been tested with two rapid tests to evidence A and B toxins. Moreover, 18 positive and 3 negative GDH samples have been examined by means of cultivation tests and using two nested PCR (n-PCR commercial kits (Neomed, Rho, Italy to amplify the CD toxin coding gene tcdC and tcdB. Results.Among 60 examined samples, 52 (45% were GDH positive, and, among these, 46 (76% and 37 (62% resulted respectively positive for both AB and for only A CD toxin using screening tests.Among the 18 GDH positive samples tested, 14 were positive for tcdC and tcdB n-PCR, while all the 3 GDH negative samples were confirmed as negative. The isolation in colture was positive in 16 of the GDH positive and in 2 of the 3 GDH negative samples. Conclusions.These data suggest that the GDH test is a useful screening method that must be associated to a confirmatory assay.The search of CD toxin coding gene by n-PCR seems to be a sensitive and specific method to assess the infection with toxins producing CD.

  20. Test structure and method for the experimental investigation of internal voltage amplification and surface potential of ferroelectric MOSFETs

    Rusu, Alexandru; Salvatore, Giovanni A.; Ionescu, Adrian M.

    2011-11-01

    In this paper we report the fabrication and detailed electrical characterization of a novel test structure based on Metal-Ferroelectric-Metal-Oxide-Semiconductor transistor with internal metal contact, aiming at extracting the surface potential and the investigation of internal voltage. This structure could possibly be used for the investigation of the differential voltage amplification expected due to negative capacitance effect. The proposed test structure is p-Fe-FET with a thin Al contact in-between the PVDF ferroelectric and a pedestal oxide, enabling access to the internal voltage potential in all the regimes of operations, from weak to strong inversion. Moreover, the capacitances of reference MOS transistor and of Fe-FET can be independently probed. The fabricated p-type Fe-FET has an excellent subthreshold slope of 75 mV/decade, Ion/Ioff > 107 and Ioff in the pA range. Based on voltage and capacitive measurements, the Fe-FET surface potential is experimentally extracted as well as the polarization of the ferroelectric layer. We demonstrate that the internal node voltage amplitude can be controlled by the sweeping conditions of the polarization loops. We propose a first order modeling of the polarization and we report simulations of the internal potential.

  1. Precision short-pulse damage test station utilizing optical parametric chirped-pulse amplification

    Jovanovic, I; Brown, C; Wattellier, B; Nielsen, N; Molander, W; Stuart, B; Pennington, D; Barty, C J

    2004-03-22

    The next generation of high-energy petawatt (HEPW)-class lasers will utilize multilayer dielectric diffraction gratings for pulse compression, due to their high efficiency and high damage threshold for picosecond pulses. The peak power of HEPW lasers will be determined by the aperture and damage threshold of the final dielectric grating in the pulse compressor and final focusing optics. We have developed a short-pulse damage test station for accurate determination of the damage threshold of the optics used on future HEPW lasers. Our damage test station is based on a highly stable, high-beam-quality optical parametric chirped-pulse amplifier (OPCPA) operating at 1053 nm at a repetition rate of 10 Hz. We present the design of our OPCPA system pumped by a commercial Q-switched pump laser and the results of the full system characterization. Initial short-pulse damage experiments in the far field using our system have been performed.

  2. Selecting Appropriate Tests to Assess the Benefits of Bilateral Amplification With Hearing Aids.

    van Schoonhoven, Jelmer; Schulte, Michael; Boymans, Monique; Wagener, Kirsten C; Dreschler, Wouter A; Kollmeier, Birger

    2016-01-01

    The aim of this study was to investigate the effect of bilateral hearing aids (HA) in subjects with mild and moderate-to-severe hearing loss. This study was designed as a within-subject feasibility study. Bilateral HA use was assessed using different laboratory tests on speech reception, listening effort, noise tolerance, and localization. All data were evaluated with bilateral and unilateral HA fittings. Forty experienced bilateral HA users were included with hearing impairment ranging from mild to moderate-to-severe. Subjects were stratified into two groups based on the degree of hearing loss. Speech reception in noise, listening effort, and localization tests showed a bilateral benefit for the moderate-to-severely hearing-impaired subjects. A bilateral benefit was also observed for listening effort in the mildly hearing-impaired group. The assessment of listening effort shows promise as a measure of bilateral HA benefit for mild hearing impairment. Localization and speech reception in noise tests provide additional value for larger losses. The next step is to compare experienced unilateral with bilateral HA users. PMID:27460871

  3. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5′ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5′ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5′ end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism. PMID:27242766

  4. Comparison of Nucleic Acid Amplification, Serology, and Microbiologic Culture for Diagnosis of Rhodococcus equi Pneumonia in Foals

    Sellon, Debra C.; Besser, Thomas E.; Vivrette, Sally L.; McConnico, Rebecca S.

    2001-01-01

    Recently, a technique was described for amplification of Rhodococcus equi-specific chromosomal and vapA DNA from blood and tracheal wash fluids. It was hypothesized that this technique would be more sensitive than standard culture techniques or serology for diagnosis of R. equi pneumonia in foals. Tracheal wash fluid, nasal swabs, whole blood samples, and serum samples from 56 foals with pneumonia were analyzed. Final clinical diagnosis was determined by the attending clinician on the basis o...

  5. Sensitivity and Specificity of Nucleic Acid Sequence-Based Amplification Method (NASBA for Diagnosis of Cutaneous Leishmaniasis

    Niazi, A

    2014-05-01

    Full Text Available Background and Objective: Employing advanced diagnostics for molecular identification of the Lishmania parasite is has a more sensitivity and specificity in comparison to the microscopic methods. RT- PCR is also introduced as one of the best known techniques for diagnosis of this parasite; however, the method is not widely used due to its expensive equipments and the time requested.the application of NASBA method is shown high efficient for diagnosis of live parasite. The aim of this study is comparison sensivity and specificity between NASBA isothermal amplification and RT-PCR for molecular detection of lishmania major. Material and Methods: 28 skin biopsy from Oscar of patients was prepared and total RNA was extracted. Then, the using of specific primers designed for 18srRNA region, this region was amplified using NASBA isothemal amplification. The RNA amplicons were produced in less than 90 minutes and then identified via electrophoresed agaros gel after staining with Syber Gold flourecent probes for the purpose increasing sensitivity and specificity Result: In this study, NASBA and RT-PCR method are sensitivity 81%, specificity of 51% and 100% respectively for detection of Leishmania parasites inscars Conclusion: NASBA isothermal method can be applied with high sensitivity and specificity for the identification of cutaneous leishmaniasis, this method can be fed with live microorganisms and response to treatment in patients examined. Keywords: Cutaneous Leishmanisis, NASBA, 18S rRNA

  6. Test-retest reliability of 10 Hz conditioning electrical stimulation inducing long-term potentiation (LTP)-like pain amplification in humans

    Xia, Weiwei; Mørch, Carsten Dahl; Andersen, Ole Kæseler

    2016-01-01

    BACKGROUND: 10 Hz conditioning electrical stimulation (CES) has been shown to induce long-term potentiation (LTP)-like pain amplification similar to traditional 100 Hz CES in healthy humans. The aim of this study was to assess the test-retest reliability and to estimate sample sizes required for...... acceptable reliability both in crossover and parallel designs (Ncr = 3, Np = 13). Pain ratings to SES were reliable, but with large estimated sample sizes (Ncr = 634, Np = 11310) due to the minor pain amplification. CONCLUSIONS: The reliability of 10 Hz CES was acceptable in inducing LTP-like effects in the...... assessments of superficial blood flow, heterotopic mechanical hyperalgesia, and dysesthesia in terms of sample sizes for future crossover study designs....

  7. Tuberculin Skin Testing

    ... American Community Summit Background Slideset Children Correctional Facilities Homelessness International Travelers Pregnancy Health Disparities Laboratory Information Model Performance Evaluation Program (MPEP) Drug Susceptibility Testing The Uses of Nucleic Acid Amplification ...

  8. Testing for TB Infection

    ... American Community Summit Background Slideset Children Correctional Facilities Homelessness International Travelers Pregnancy Health Disparities Laboratory Information Model Performance Evaluation Program (MPEP) Drug Susceptibility Testing The Uses of Nucleic Acid Amplification ...

  9. SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

    Ricardo Luiz Dantas MACHADO

    1998-09-01

    Full Text Available We report an adaptation of a technique for the blood sample collection (GFM as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.Relatamos a adaptação de uma técnica para coleta de amostras (MFV e outra para extração, amplificação de DNA de parasitas da malária para diagnóstico por PCR/ELISA. O método de coleta de amostras requer menos habilidade e economisa tempo e dinheiro, assim reduzindo a mais da metade o custo. O material é também adequado para análise genética em especimens frescos ou estocados, preparados por este método.

  10. Application of an in vitro-amplification assay as a novel pre-screening test for compounds inhibiting the aggregation of prion protein scrapie

    Matthias Schmitz; Maria Cramm; Franc Llorens; Niccolò Candelise; Dominik Müller-Cramm; Daniela Varges; Schulz-Schaeffer, Walter J.; Saima Zafar; Inga Zerr

    2016-01-01

    In vitro amplification assays, such as real-time quaking-induced conversion (RT-QuIC) are used to detect aggregation activity of misfolded prion protein (PrP) in brain, cerebrospinal fluid (CSF) and urine samples from patients with a prion disease. We believe that the method also has a much broader application spectrum. In the present study, we applied RT-QuIC as a pre-screening test for substances that potentially inhibit the aggregation process of the cellular PrP (PrPC) to proteinase (PK)-...

  11. A label-free fluorescent probe based on DNA-templated silver nanoclusters and exonuclease III-assisted recycling amplification detection of nucleic acid.

    Yang, Wen; Tian, Jianniao; Ma, Yefei; Wang, Lijun; Zhao, Yanchun; Zhao, Shulin

    2015-11-01

    A number of specific nucleic acids are closely related with many serious diseases, in the current research, a platform taking advantage of exonuclease III (Exo III) to realize double recycling amplification and label-free fluorescent DNA-templated silver nanoclusters (DNA-AgNCs) for detecting of nucleic acid had been developed. In this method, a molecular beacon (MB) with 3'-protruding termini and a single-stranded cytosine-rich (C-rich) probe were designed that coexist stably with Exo III. Once the target DNA appeared, portion of the MB could hybridize with target DNA and was digested by Exo III, which allowed the release of target DNA and a residual sequence. Subsequently, the residual sequence could trigger the Exo III to digest C-rich probe, and the DNA-AgNCs was not able to be synthesized because of the C-rich probe was destroyed; finally the fluorescent of solution was quenched. This assay enables to monitor human hemochromatosis gene (as a model) with high sensitivity, the detection limit is as low as 120 pM compared with other fluorescence DNA-AgNCs methods, this assay also exhibits superior specificity even against single base mismatch. The strategy is applied to detect human hemochromatosis gene in real human serum samples successfully. PMID:26572843

  12. Amplification-Free Detection of Circulating microRNA Biomarkers from Body Fluids Based on Fluorogenic Oligonucleotide-Templated Reaction between Engineered Peptide Nucleic Acid Probes: Application to Prostate Cancer Diagnosis.

    Metcalf, Gavin A D; Shibakawa, Akifumi; Patel, Hinesh; Sita-Lumsden, Ailsa; Zivi, Andrea; Rama, Nona; Bevan, Charlotte L; Ladame, Sylvain

    2016-08-16

    Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening. PMID:27498854

  13. Isothermal DNA amplification in vitro: the helicase-dependent amplification system.

    Jeong, Yong-Joo; Park, Kkothanahreum; Kim, Dong-Eun

    2009-10-01

    Since the development of polymerase chain reaction, amplification of nucleic acids has emerged as an elemental tool for molecular biology, genomics, and biotechnology. Amplification methods often use temperature cycling to exponentially amplify nucleic acids; however, isothermal amplification methods have also been developed, which do not require heating the double-stranded nucleic acid to dissociate the synthesized products from templates. Among the several methods used for isothermal DNA amplification, the helicase-dependent amplification (HDA) is discussed in this review with an emphasis on the reconstituted DNA replication system. Since DNA helicase can unwind the double-stranded DNA without the need for heating, the HDA system provides a very useful tool to amplify DNA in vitro under isothermal conditions with a simplified reaction scheme. This review describes components and detailed aspects of current HDA systems using Escherichia coli UvrD helicase and T7 bacteriophage gp4 helicase with consideration of the processivity and efficiency of DNA amplification. PMID:19629390

  14. Selective Adsorption and Chiral Amplification of Amino Acids in Vermiculite Clay -Implications for the origin of biochirality

    Fraser, Donald G; Jakschitz, Thomas; Rode, Bernd M

    2010-01-01

    Smectite clays are hydrated layer silicates that, like micas, occur naturally in abundance. Importantly, they have readily modifiable interlayer spaces that provide excellent sites for nanochemistry. Vermiculite is one such smectite clay and in the presence of small chain-length alkyl-NH3Cl ions, forms sensitive, 1-D ordered model clay systems with expandable nano-pore inter-layer regions. These inter-layers readily adsorb organic molecules. N-propyl NH3Cl vermiculite clay gels were used to determine the adsorption of alanine, lysine and histidine by chiral HPLC. The results show that during reaction with fresh vermiculite interlayers, significant chiral enrichment of either L- and D-enantiomers occurs depending on the amino acid. Chiral enrichment of the supernatant solutions is up to about 1% per pass. In contrast, addition to clay interlayers already reacted with amino acid solutions resulted in little or no change in D/L ratio during the time of the experiment. Adsorption of small amounts of amphiphilic o...

  15. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

    P.F. Mens; G.J. Schoone; P.A. Kager; H.D.F.H. Schallig

    2006-01-01

    Background: Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (< 20 parasites/mu l) the technique becomes less sensitive and time consuming. Rapid diagnostic tests based on Plasmodium antigen detection

  16. Detection of Aspergillus fumigatus in a rat model of invasive pulmonary aspergillosis by real-time nucleic acid sequence-based amplification.

    Zhao, Yanan; Park, Steven; Warn, Peter; Shrief, Raghdaa; Harrison, Elizabeth; Perlin, David S

    2010-04-01

    Rapid and sensitive detection of Aspergillus from clinical samples may facilitate the early diagnosis of invasive pulmonary aspergillosis (IPA). A real-time nucleic acid sequence-based amplification (NASBA) method was investigated by use of an inhalational rat model of IPA. Immunosuppressed male Sprague-Dawley rats were exposed to Aspergillus fumigatus spores for an hour in an aerosol chamber. Bronchoalveolar lavage (BAL) fluid, lung tissues, and whole blood were collected from five infected rats at 1, 24, 48, 72, and 96 h postinfection and five uninfected rats at the end of the experiment. Total nucleic acid (TNA) was extracted on an easyMAG instrument. A primer-molecular beacon set targeting 28S rRNA was designed to detect Aspergillus spp. The results were compared to those of quantitative PCR (qPCR) (18S rDNA) and quantitative culture. The analytical sensitivity of the real-time NASBA assay was tissue burdens, while the CFU counts were stable over time. The fungal burdens in BAL fluid were more variable and not indicative of a progressive infection. The results of both real-time assays correlated well for both sample types (r = 0.869 and P tissue, r = 0.887 and P < 0.0001 for BAL fluid). For all whole-blood specimens, NASBA identified Aspergillus-positive samples in the group from which samples were collected at 72 h postinfection (three of five samples) and the group from which samples were collected at 96 h postinfection (five of five samples), but no positive results were obtained by culture or PCR. Real-time NASBA is highly sensitive and useful for the detection of Aspergillus in an experimental model of IPA. PMID:20129972

  17. Simple system for isothermal DNA amplification coupled to lateral flow detection.

    Kristina Roskos

    Full Text Available Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP or the Exponential Amplification Reaction (EXPAR, both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.

  18. Cost analysis of nucleic acid amplification for diagnosing pulmonary tuberculosis, within the context of the Brazilian Unified Health Care System

    Márcia Pinto

    2015-12-01

    Full Text Available ABSTRACT We estimated the costs of a molecular test for Mycobacterium tuberculosis and resistance to rifampin (Xpert MTB/RIF and of smear microscopy, within the Brazilian Sistema Único de Saúde (SUS, Unified Health Care System. In SUS laboratories in the cities of Rio de Janeiro and Manaus, we performed activity-based costing and micro-costing. The mean unit costs for Xpert MTB/RIF and smear microscopy were R$35.57 and R$14.16, respectively. The major cost drivers for Xpert MTB/RIF and smear microscopy were consumables/reagents and staff, respectively. These results might facilitate future cost-effectiveness studies and inform the decision-making process regarding the expansion of Xpert MTB/RIF use in Brazil.

  19. Evaluation of a viral microarray based on simultaneous extraction and amplification of viral nucleotide acid for detecting human herpesviruses and enteroviruses.

    Yi Liu

    Full Text Available In this study, a viral microarray based assay was developed to detect the human herpesviruses and enteroviruses associated with central nervous system infections, including herpes simplex virus type 1, type 2 (HSV1 and HSV2, Epstein-Barr virus (EBV, cytomegalovirus (CMV, enterovirus 71 (EV71, coxsackievirus A 16 (CA16 and B 5(CB5. The DNA polymerase gene of human herpesviruses and 5'-untranslated region of enteroviruses were selected as the targets to design primers and probes. Human herpesviruses DNA and enteroviruses RNA were extracted simultaneously by using a guanidinium thiocyanate acid buffer, and were subsequently amplified through a biotinylated asymmetry multiplex RT-PCR with the specific primer of enteroviruses. In total, 90 blood samples and 49 cerebrospinal fluids samples with suspected systemic or neurological virus infections were investigated. Out of 139 samples, 66 were identified as positive. The specificities of this multiplex RT-PCR microarray assay were over 96% but the sensitivities were various from 100% for HSV1, HSV2, EV71 and CB5, 95.83% for CMV, 80% for EBV to 71.43% for CA16 in comparison with reference standards of TaqMan qPCR/qRT-PCR. The high Kappa values (>0.90 from HSV1, HSV2, CMV, EV71 and CB5 were obtained, indicating almost perfect agreement in term of the 5 viruses detection. But lower Kappa values for EBV (0.63 and CA16 (0.74 displayed a moderate to substantial agreement. This study provides an innovation of simultaneous extraction, amplification, hybridization and detection of DNA viruses and RNA viruses with simplicity and specificity, and demonstrates a potential clinical utility for a variety of viruses' detection.

  20. 21 CFR 862.1320 - Gastric acidity test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Gastric acidity test system. 862.1320 Section 862.1320 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  1. 21 CFR 862.1290 - Fatty acids test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fatty acids test system. 862.1290 Section 862.1290 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  2. 21 CFR 862.1095 - Ascorbic acid test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Ascorbic acid test system. 862.1095 Section 862.1095 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  3. 21 CFR 862.1295 - Folic acid test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Folic acid test system. 862.1295 Section 862.1295 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  4. Tested Demonstrations: Color Oscillations in the Formic Acid-Nitric Acid-Sulfuric Acid System.

    Raw, C. J. G.; And Others

    1983-01-01

    Presented are procedures for demonstrating the production of color oscillations when nitric acid is added to a formic acid/concentrated sulfuric acid mixture. Because of safety considerations, "Super-8" home movie of the color changes was found to be satisfactory for demonstration purposes. (JN)

  5. An integrated lateral flow assay for effective DNA amplification and detection at the point of care.

    Choi, Jane Ru; Hu, Jie; Gong, Yan; Feng, Shangsheng; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

    2016-05-10

    Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future. PMID:27010033

  6. Molecular staging of lymph node-negative colon carcinomas by one-step nucleic acid amplification (OSNA) results in upstaging of a quarter of patients in a prospective, European, multicentre study

    Croner, R.S.; Geppert, C-I; Bader, F G; Nitsche, U.; Späth, C; Rosenberg, R.; Zettl, A.; Matias-Guiu, X; Tarragona, J; Güller, U.; Stürzl, M; Zuber, M

    2014-01-01

    Background: Current histopathological staging procedures in colon carcinomas depend on midline division of the lymph nodes with one section of haematoxylin & eosin (H&E) staining only. By this method, tumour deposits outside this transection line may be missed and could lead to understaging of a high-risk group of stage UICC II cases, which recurs in ∼20% of cases. A new diagnostic semiautomated system, one-step nucleic acid amplification (OSNA), detects cytokeratin (CK) 19 mRNA in lymph node...

  7. 21 CFR 862.3580 - Lysergic acid diethylamide (LSD) test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lysergic acid diethylamide (LSD) test system. 862... Test Systems § 862.3580 Lysergic acid diethylamide (LSD) test system. (a) Identification. A lysergic acid diethylamide (LSD) test system is a device intended to measure lysergic acid diethylamide,...

  8. Evaluation of Three Automated Nucleic Acid Amplification Systems for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in First-Void Urine Specimens▿

    Levett, P N; Brandt, K.; Olenius, K.; Brown, C.; Montgomery, K.; Horsman, G. B.

    2008-01-01

    A total of 500 first-void urine specimens were tested for the presence of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids using ProbeTec ET reagents on a Viper platform (BD Diagnostics, Mississauga, Ontario, Canada), Aptima Combo 2 reagents on a Tigris platform (Gen-Probe, Inc., San Diego, CA), and Abbott RealTime CT/NG reagents on an m2000 platform (Abbott Molecular Diagnostics, Des Plaines, IL). The performance of the three assays for detection of N. gonorrhoeae was comparable...

  9. Standing of nucleic acid testing strategies in veterinary diagnosis laboratories to uncover Mycobacterium tuberculosis complex members.

    Costa, Pedro; Botelho, Ana; Couto, Isabel; Viveiros, Miguel; Inácio, João

    2014-01-01

    Nucleic acid testing (NAT) designate any molecular approach used for the detection, identification, and characterization of pathogenic microorganisms, enabling the rapid, specific, and sensitive diagnostic of infectious diseases, such as tuberculosis. These assays have been widely used since the 90s of the last century in human clinical laboratories and, subsequently, also in veterinary diagnostics. Most NAT strategies are based in the polymerase chain reaction (PCR) and its several enhancements and variations. From the conventional PCR, real-time PCR and its combinations, isothermal DNA amplification, to the nanotechnologies, here we review how the NAT assays have been applied to decipher if and which member of the Mycobacterium tuberculosis complex is present in a clinical sample. Recent advances in DNA sequencing also brought new challenges and have made possible to generate rapidly and at a low cost, large amounts of sequence data. This revolution with the high-throughput sequencing (HTS) technologies makes whole genome sequencing (WGS) and metagenomics the trendiest NAT strategies, today. The ranking of NAT techniques in the field of clinical diagnostics is rising, and we provide a SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis with our view of the use of molecular diagnostics for detecting tuberculosis in veterinary laboratories, notwithstanding the gold standard being still the classical culture of the agent. The complementary use of both classical and molecular diagnostics approaches is recommended to speed the diagnostic, enabling a fast decision by competent authorities and rapid tackling of the disease. PMID:25988157

  10. K Basin Sludge Conditioning Testing: Nitric Acid Dissolution Testing of K East Canister Sludge

    This report describes tests performed by Pacific Northwest National Laboratory (PNNL) for Numatec Hanford Corporation (NHC) as part of the overall activities for the development of the K Basin Sludge Treatment System. These tests were conducted to examine the dissolution behavior of a K East Basin canister sludge composite in nitric acid at the following concentrations: 2 M, 4 M, 6 M, 7.8 M and 10 M and temperatures of 25 C and boiling. Assuming that the sludge was 100% uranium metal, a 4X stoichiometric excess of nitric acid was used for all testing, except that conducted at 4 M. In the 4 M nitric acid dissolution test, 50% excess nitric acid was used resulting in a dissolver solution with a significantly higher solids loading. The boiling tests were conducted for 11 hr, the 25 C dissolution tests were conducted from 24 hr to 2 weeks. For the 25 C dissolution testing, the weight percent residual solids was determined, however, chemical and radiochemical analyses were not performed

  11. Comparison of Two Amplification Technologies for Detection and Quantitation of Human Immunodeficiency Virus Type 1 RNA in the Female Genital Tract

    Bremer, James; Nowicki, Marek; Beckner, Suzanne; Brambilla, Donald; Cronin, Mike; Herman, Steven; Kovacs, Andrea; Reichelderfer, Patricia

    2000-01-01

    Human immunodeficiency virus type 1 (HIV-1) RNA levels in female genital tract and peripheral blood samples were compared using two commercial amplification technologies: the Roche AMPLICOR HIV-1 MONITOR test and either the Organon Teknika nucleic acid sequence-based amplification (NASBA-QT) assay or the NucliSens assay. Estimates of HIV-1 RNA copy number were derived from internal kit standards and analyzed unadjusted and adjusted to a common set of external standards. We found a discordance...

  12. Operation of the radioactive acid-digestion test unit (RADTU)

    At the Karlsruhe Nuclear Research Center, research and development work concerned with the acid digestion of combustible waste with high plutonium content is being carried out. The main objectives are the reduction of the waste volume, the recovery of the plutonium and the transformation of the process residues into a product suitable for final disposal. For this purpose an inactive demonstration plant with a throughput of 1.5 kg/h has been constructed. In the first version, the reactor of this plant was a tray reactor of the HEDL type. During test operations the sedimentation of a residue was observed on the tray. The density of this residue was higher than that of the residue suspended in the reaction acid. Experiments using tantalum oxide (D=8.4 g/cm3) to simulate the plutonium oxide (D=11.5 g/cm3) have verified this observation. This means that in active operation a sedimentation of plutonium oxide on the tray is to be expected. With respect to these results a new reactor was developed where the settling of the residue is avoided by appropriate design and high circulation velocity of the reaction acid. The reactor consists of a tube 50-80 mm in diameter, which is curved to form a closed loop with an arm length of about 1x1 m. Since October 1979 this ring reactor has been under inactive test operation. Its behaviour is very stable. The velocity of the reaction acid in the heater is 0.5-1 m/s. A settling of residues or tantalum oxide has not been observed. The throughput attained is comparable to that of the tray reactor but the space requirement is three times smaller, and the volume of the reaction acid four times smaller. (author)

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

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

    2016-05-15

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

  14. Amplification for the Adolescent.

    Wilber, Laura Ann

    1978-01-01

    Explored are various means of amplification for aurally handicapped adolescents, including behind-the-ear hearing aids, "custom ear" (or in-the-ear) hearing aids, as well as aural rehabilitation. (BD)

  15. Early amplification options.

    Gabbard, Sandra Abbott; Schryer, Jennifer

    2003-01-01

    Children with permanent hearing loss have been remediated with hearing amplification devices for decades. The influx of young infants identified with hearing loss through successful newborn hearing screening programs has established a need for amplification resources for infants within the first six months of life. For the approximately two of every 1000 infants born who are identified with bilateral hearing loss [Mehl and Thomson, 1998, Pediatrics 101, p. e4], the use of amplification is commonly the first step in treating the sequella of their loss. The use of hearing aids, combined with early intervention, has been shown to significantly improve the speech and language skills of young children with hearing loss [Yoshinaga-Itano, 2000, Seminars in Hearing 21, p. 309]. Speech and language delays have contributed to compromised academic performance of school aged children with hearing loss [Johnson et al., 1997, Educational Audiology Handbook, Singular Publishing, San Diego]. Most hard-of-hearing and deaf children use hearing aids and other assistive listening devices every day throughout their lifetime and the life expectancy of a hearing aid is only five to eight years. The current challenge for pediatric audiologists is selecting and evaluating the available amplification to provide the best options for children and their families. Amplification technology has seen an explosion in growth the past few years and the options continue to expand rapidly. This article examines currently available amplification technology and reviews the selection criteria that may be used for infants and young children. Issues such as style, type, amplification features, signal processing strategies, and verification and validation tools are also discussed. PMID:14648816

  16. 21 CFR 862.1187 - Conjugated sulfolithocholic acid (SLCG) test system.

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1187 Conjugated sulfolithocholic acid (SLCG) test system. (a) Identification....

  17. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

    David S Boyle

    Full Text Available Improved access to effective tests for diagnosing tuberculosis (TB has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110 and 20 fg (IS1081were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9 and 86.1% (95%CI: 78.1, 94.1 respectively (n = 71. Specificities were 100% and 88.6% (95% CI: 80.8, 96.1 respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2 and 70.8% (95%CI: 62.9, 78.7 were obtained (n = 90. Specificities were 95.4 (95% CI: 92.3,98.1 and 88% (95% CI: 83.6, 92.4 respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB

  18. Polymerase chain reaction amplification of a GC rich region by adding 1,2 propanediol

    Zeinab Mousavian

    2014-01-01

    Full Text Available Background : Apolipoprotein E (ApoE is one of the most important carriers of lipids in mammalians. The gene for this lipoprotein (ApoE is located on chromosome 19 which is related with the pathogenesis of some nervous system disease. ApoE gene is identified as a high guanine-cytosine (GC content fragment. Detection and amplification of these templates are extensively laborious and baffling. The aim of this study was to find a practical and feasible method for the amplification of the number of GC rich genes such as ApoE. Materials and Methods: We experimented with simple polymerase chain reaction (PCR, nested PCR and PCR with 1-2 propanediol, dimethylsulfoxide (DMSO, and ethyleneglicol as additive substances to enhance the amplification ApoE gene and used the 40 samples of the human whole blood were collected in test tubes with a pre-treatment of ethylene diaminetetraacetic acid. Results: According to our observations, presence of 1-2 propanediol, DMSO, and ethyleneglicol as additive substances resulted to enhanced amplification of ApoE gene. Addition of 1-2 propanediol showed the best results, caused optimization and revealed more specific and sharp bands. Conclusion: According to our findings 1-2 propanediol are the best organic reagent for improving the amplification of ApoE gene. Optimization procedure for each GC rich sequence is recommended to be performed separately in order to identify which of the additive agent is more efficient and applicable for a particular target.

  19. Single genome amplification and direct amplicon sequencing of Plasmodium spp. DNA from ape fecal specimens

    Liu, Weimin; Li, Yingying; Peeters, Martine; Rayner, Julian; Sharp, Paul; Shaw, George; Hahn, Beatrice

    2010-01-01

    Conventional PCR followed by molecular cloning and sequencing of amplified products is commonly used to test clinical specimens for target sequences of interest, such as viral, bacterial or parasite nucleic acids. However, this approach has serious limitations when used to analyze mixtures of genetically divergent templates1–9. This is because Taq polymerase is prone to switch templates during the amplification process, thereby generating recombinants that do not exist in vivo4. When amplicon...

  20. Single genome amplification and direct amplicon sequencing of Plasmodium spp. DNA from ape fecal specimens

    sprotocols

    2015-01-01

    Conventional PCR followed by molecular cloning and sequencing of amplified products is commonly used to test clinical specimens for target sequences of interest, such as viral, bacterial or parasite nucleic acids. However, this approach has serious limitations when used to analyze mixtures of genetically divergent templates (1-9). This is because Taq polymerase is prone to switch templates during the amplification process, thereby generating recombinants that do not exist in vivo (4). When am...

  1. Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing.

    Trösemeier, Jan-Hendrik; Musso, Didier; Blümel, Johannes; Thézé, Julien; Pybus, Oliver G; Baylis, Sally A

    2016-01-01

    We report here the sequence of a candidate reference strain of Zika virus (ZIKV) developed on behalf of the World Health Organization (WHO). The ZIKV reference strain is intended for use in nucleic acid amplification (NAT)-based assays for the detection and quantification of ZIKV RNA. PMID:27587826

  2. Study of the population dynamics of a mixed bacterial culture able to degrade cyanuric acid in a packed bed reactor, using RAPD (Random amplification of polymorphic DNA) technique

    Cyanuric acid is a biodegradation byproduct of triazinic compounds. Because of its low carbon to nitrogen ratio, a complementary carbon source is usually needed for its complete biodegradation. In this work, glucose was used as extra carbon source. Cyanuric hydrolase is the first enzyme in cyanuric acid (CA) catabolism, and is produced by a wide number of microorganisms. (Author)

  3. Quantum Feedback Amplification

    Yamamoto, Naoki

    2016-04-01

    Quantum amplification is essential for various quantum technologies such as communication and weak-signal detection. However, its practical use is still limited due to inevitable device fragility that brings about distortion in the output signal or state. This paper presents a general theory that solves this critical issue. The key idea is simple and easy to implement: just a passive feedback of the amplifier's auxiliary mode, which is usually thrown away. In fact, this scheme makes the controlled amplifier significantly robust, and furthermore it realizes the minimum-noise amplification even under realistic imperfections. Hence, the presented theory enables the quantum amplification to be implemented at a practical level. Also, a nondegenerate parametric amplifier subjected to a special detuning is proposed to show that, additionally, it has a broadband nature.

  4. Heralded amplification of photonic qubits.

    Bruno, Natalia; Pini, Vittorio; Martin, Anthony; Verma, Varun B; Nam, Sae Woo; Mirin, Richard; Lita, Adriana; Marsili, Francesco; Korzh, Boris; Bussières, Félix; Sangouard, Nicolas; Zbinden, Hugo; Gisin, Nicolas; Thew, Rob

    2016-01-11

    We demonstrate postselection free heralded qubit amplification for Time-Bin qubits and single photon states in an all-fibre, telecom-wavelength, scheme that highlights the simplicity, stability and potential for fully integrated photonic solutions. Exploiting high-efficiency superconducting detectors, the gain, fidelity and the performance of the amplifier are studied as a function of loss. We also demonstrate the first heralded single photon amplifier with independent sources. This provides a significant advance towards demonstrating device-independent quantum key distribution as well as fundamental tests of quantum mechanics over extended distances. PMID:26832244

  5. Label-free and ratiometric detection of nuclei acids based on graphene quantum dots utilizing cascade amplification by nicking endonuclease and catalytic G-quadruplex DNAzyme.

    Wang, Guang-Li; Fang, Xin; Wu, Xiu-Ming; Hu, Xue-Lian; Li, Zai-Jun

    2016-07-15

    Herein, we report a ratiometric fluorescence assay based on graphene quantum dots (GQDs) for the ultrasensitive DNA detection by coupling the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for cascade signal amplifications. With o-phenylenediamine acted as the substrate of G-quadruplex/hemin DNAzyme, whose oxidization product (that is, 2,3-diaminophenazine, DAP) quenched the fluorescence intensity of GQDs (at 460nm) obviously, accompanied with the emergence of a new emission of DAP (at 564nm). The ratiometric signal variations at the emission wavelengths of 564 and 460nm (I564/I460) were utilized for label-free, sensitive, and selective detection of target DNA. Utilizing the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for amplified cascade generation of DAP, the proposed bioassay exhibited high sensitivity toward target DNA with a detection limit of 30fM. The method also had additional advantages such as facile preparation and easy operation. PMID:26950646

  6. On soliton amplification

    Leibovich, S.; Randall, J. D.

    1979-01-01

    The paper considers a modified Korteweg-de Vries equation that permits wave amplification or damping. A 'terminal similarity' solution is identified for large times in amplified systems. Numerical results are given which confirm that the terminal similarity solution is a valid local approximation for mu t sufficiently large and positive, even though the approximation is not uniformly valid in space.

  7. Chlamydia Testing

    ... Amplification Test (NAAT); Chlamydia trachomatis Culture; Chlamydia trachomatis DNA Probe Related tests: Gonorrhea Testing , HIV Antibody and HIV Antigen , Syphilis Tests , Herpes Testing , HPV Test , Trichomonas Testing All content on Lab Tests Online has ...

  8. Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

    Rutjes, Saskia A.; van den Berg, Harold H. J. L.; Lodder, Willemijn J.; Roda Husman, Ana Maria de

    2006-01-01

    Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performa...

  9. Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay.

    Rutjes, Saskia A.; Berg, Harold H J L van den; Lodder, Willemijn J.; Roda Husman, Ana Maria de

    2006-01-01

    Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performa...

  10. Acid Pit Stabilization Project (Volume 1 - Cold Testing) and (Volume 2 - Hot Testing)

    During the summer and fall of Fiscal Year 1997, a Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) Treatability Study was performed at the Idaho National Engineering and Environmental Laboratory. The study involved subsurface stabilization of a mixed waste contaminated soil site called the Acid Pit. This study represents the culmination of a successful technology development effort that spanned Fiscal Years 1994-1996. Research and development of the in situ grout stabilization technique was conducted. Hardware and implementation techniques are currently documented in a patent pending with the United States Patent and Trademark Office. The stabilization technique involved using jet grouting of an innovative grouting material to form a monolith out of the contamination zone. The monolith simultaneously provides a barrier to further contaminant migration and closes voids in the soil structure against further subsidence. This is accomplished by chemical incorporation of contaminants into less soluble species and achieving a general reduction in hydraulic conductivity within the monolith. The grout used for this study was TECT-HG, a relatively dense iron oxide-based cementitious grout. The treatability study involved cold testing followed by in situ stabilization of the Acid Pit. Volume 1 of this report discusses cold testing, performed as part of a ''Management Readiness Assessment'' in preparation for going hot. Volume 2 discusses the results of the hot Acid Pit Stabilization phase of this project. Drilling equipment was specifically rigged to reduce the spread of contamination, and all grouting was performed under a concrete block containing void space to absorb any grout returns. Data evaluation included examination of implementability of the grouting process and an evaluation of the contaminant spread during grouting. Following curing of the stabilized pit, cores were obtained and evaluated for toxicity characteristic leach ing

  11. Improved PCR Amplification of Broad Spectrum GC DNA Templates

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10–90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content. PMID:27271574

  12. Amplification of an MFS Transporter Encoding Gene penT Significantly Stimulates Penicillin Production and Enhances the Sensitivity of Penicillium chrysogenum to Phenylacetic Acid

    Jing Yang; Xinxin Xu; Gang Liu

    2012-01-01

    Penicillin is historically important as the first discovered drug against bacterial infections in human.Although the penicillin biosynthetic pathway and regulatory mechanism have been well studied in Penicillium chrysogenum,the compartnentation and molecular transport of penicillin or its precursors are still poorly understood.In search of the genomic database,more than 830 open reading frames (ORFs) were found to encode transmembrane proteins of P.chrysogenum.In order to investigate their roles on penicillin production,one of them (penT) was selected and cloned.The deduced protein of penT belongs to the major facilitator superfamily (MFS) and contains 12transmembrane spanning domains (TMS).During fermentation,the transcription of penT was greatly induced by penicillin precursors phenylacetic acid (PAA) and phenoxyacetic acid (POA).Knock-down of penT resulted in significant decrease of penicillin production,while over-expression of penT under the promoter of trpC enhanced the penicillin production.Introduction of an additional penT in the wild-type strain of P.chrysogenum doubled the penicillin production and enhanced the sensitivity of P.chrysogenum to the penicillin precursors PAA or POA.These results indicate that penT stimulates penicillin production probably through enhancing the translocation of penicillin precursors across fungal cellular membrane.

  13. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-05-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease. PMID:27284221

  14. Loop-Mediated Amplification Accelerated by Stem Primers

    Laurence Tisi; Guy Kiddle; Olga Gandelman; Rebecca Jackson

    2011-01-01

    Isothermal nucleic acid amplifications (iNAATs) have become an important alternative to PCR for in vitro molecular diagnostics in all fields. Amongst iNAATs Loop-mediated amplification (LAMP) has gained much attention over the last decade because of the simplicity of hardware requirements. LAMP demonstrates performance equivalent to that of PCR, but its application has been limited by the challenging primer design. The design of six primers in LAMP requires a selection of eight priming sites ...

  15. The role of boric acid in the Phebus-FP tests

    The data in this report should enable proposed methods for the introduction of boric acid into the Phebus-FP tests to be assessed. Boric acid chemistry is reviewed in the condensed, vapour and aqueous phases. The chemistry of concentrated aqueous solutions of boric acid is complex, involving polyborate species resulting in enhanced acidities. Equations are presented that allow the partition of boric acid between the vapour and aqueous phase to be calculated at different boric acid concentrations. The volatility of boric acid is enhanced significantly in the presence of steam. Equations are given that allow the effective vapour pressure of boric acid to be determined as functions of the water vapour pressure for temperatures in the range from 723 to 1000oC. These data are confirmed by experimental studies designed to simulate specific aspects of the conditions in the Phebus-FP tests. (author)

  16. Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

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

    2014-01-01

    Background Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species. Methodology/Principal Findings For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. Conclusions/Significance The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests. PMID:25379890

  17. Acid Pit Stabilization Project (Volume 1 - Cold Testing) and (Volume 2 - Hot Testing)

    G. G. Loomis (INEEL); A. P. Zdinak (MSE); M. A. Ewanic (MSE); J. J. Jessmore (INEEL)

    1998-01-01

    During the summer and fall of Fiscal Year 1997, a Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) Treatability Study was performed at the Idaho National Engineering and Environmental Laboratory. The study involved subsurface stabilization of a mixed waste contaminated soil site called the Acid Pit. This study represents the culmination of a successful technology development effort that spanned Fiscal Years 1994-1996. Research and development of the in situ grout stabilization technique was conducted. Hardware and implementation techniques are currently documented in a patent pending with the United States Patent and Trademark Office. The stabilization technique involved using jet grouting of an innovative grouting material to form a monolith out of the contamination zone. The monolith simultaneously provides a barrier to further contaminant migration and closes voids in the soil structure against further subsidence. This is accomplished by chemical incorporation of contaminants into less soluble species and achieving a general reduction in hydraulic conductivity within the monolith. The grout used for this study was TECT-HG, a relatively dense iron oxide-based cementitious grout. The treatability study involved cold testing followed by in situ stabilization of the Acid Pit. Volume 1 of this report discusses cold testing, performed as part of a ''Management Readiness Assessment'' in preparation for going hot. Volume 2 discusses the results of the hot Acid Pit Stabilization phase of this project. Drilling equipment was specifically rigged to reduce the spread of contamination, and all grouting was performed under a concrete block containing void space to absorb any grout returns. Data evaluation included examination of implementability of the grouting process and an evaluation of the contaminant spread during grouting. Following curing of the stabilized pit, cores were obtained and evaluated for toxicity

  18. Rapid Diagnosis of Human Herpesvirus 6 Infection by a Novel DNA Amplification Method, Loop-Mediated Isothermal Amplification

    Ihira, Masaru; Yoshikawa, Tetsushi; Enomoto, Yoshihiko; Akimoto, Shiho; Ohashi, Masahiro; Suga, Sadao; Nishimura, Naoko; Ozaki, Takao; Nishiyama, Yukihiro; Notomi, Tsugunori; Ohta, Yoshinori; Asano, Yoshizo

    2004-01-01

    A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The s...

  19. Nucleic acid tests for the detection of alpha human papillomaviruses.

    Poljak, Mario; Cuzick, Jack; Kocjan, Boštjan J; Iftner, Thomas; Dillner, Joakim; Arbyn, Marc

    2012-11-20

    Testing for high-risk types of alpha human papillomaviruses (HPV) is an invaluable part of clinical guidelines for cervical carcinoma screening, management and treatment. In this comprehensive inventory of commercial tests for detection of alpha-HPV, we identified at least 125 distinct HPV tests and at least 84 variants of the original tests. However, only a small subset of HPV tests has documented clinical performance for any of the standard HPV testing indications. For more than 75% of HPV tests currently on the market, no single publication in peer-reviewed literature can be identified. HPV tests that have not been validated and lack proof of reliability, reproducibility and accuracy should not be used in clinical management. Once incorporated in the lab, it is essential that the whole procedure of HPV testing is subject to continuous and rigorous quality assurance to avoid sub-optimal, potentially harmful practices. Manufacturers of HPV tests are urged to put more effort into evaluating their current and future products analytically, using international standards, and for clinical applications, using clinically validated endpoints. To assist with analytical validation, the World Health Organization is developing international standards for HPV types other than HPV16 and HPV18 and is planning development of external quality control panels specifically designed to be used for performance evaluation of current and future HPV tests. There is a need for more competitively priced HPV tests, especially for resource-poor countries, and uniform test validation criteria based on international standards should enable issuing more competitive and fair tender notices for purchasing. Automation systems allowing large-scale testing, as well as further increases in clinical performance, are the main needs in the further improvement of HPV tests. This article forms part of a special supplement entitled "Comprehensive Control of HPV Infections and Related Diseases" Vaccine

  20. Nitric acid flowsheet with late wash PHA testing

    This Task Technical Plan outlines the activities to be conducted in the Integrated DWPF Melter System (IDMS) in ongoing support of the Defense Waste Processing Facility (DWPF) Chemical Process Cell (CPC) utilizing the Nitric Acid Flowsheet in the Sludge Receipt and Adjustment Tank (SRAT) and Precipitate Hydrolysis Aqueous (PHA) produced by the Late Wash Flowsheet. The IDMS facility is to be operated over a series of runs (2 to 4) using the Nitric Acid Flowsheet. The PHA will be produced with the Late Wash Flowsheet in the Precipitate Hydrolysis Experimental Facility (PHEF). All operating conditions shall simulate the expected DWPF operating conditions as closely as possible. The task objectives are to perform at least two IDMS runs with as many operating conditions as possible at nominal DWPF conditions. The major purposes of these runs are twofold: verify that the combined Late Wash and Nitric Acid flowsheets produce glass of acceptable quality without additional changes to process equipment, and determine the reproducibility of data from run to run. These runs at nominal conditions will be compared to previous runs made with PHA produced from the Late Wash flowsheet and with the Nitric Acid flowsheet in the SRAT (Purex 4 and Purex 5)

  1. Evidence of high-elevation amplification versus Arctic amplification

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-01

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

  2. An Optical Test Strip for the Detection of Benzoic Acid in Food

    Fatimah Abu Bakar

    2011-07-01

    Full Text Available Fabrication of a test strip for detection of benzoic acid was successfully implemented by immobilizing tyrosinase, phenol and 3-methyl-2-benzothiazolinone hydrazone (MBTH onto filter paper using polystyrene as polymeric support. The sensing scheme was based on the decreasing intensity of the maroon colour of the test strip when introduced into benzoic acid solution. The test strip was characterized using optical fiber reflectance and has maximum reflectance at 375 nm. It has shown a highly reproducible measurement of benzoic acid with a calculated RSD of 0.47% (n = 10. The detection was optimized at pH 7. A linear response of the biosensor was obtained in 100 to 700 ppm of benzoic acid with a detection limit (LOD of 73.6 ppm. At 1:1 ratio of benzoic acid to interfering substances, the main interfering substance is boric acid. The kinetic analyses show that, the inhibition of benzoic is competitive inhibitor and the inhibition constant (Ki is 52.9 ppm. The activity of immobilized tyrosinase, phenol, and MBTH in the test strip was fairly sustained during 20 days when stored at 3 °C. The developed test strip was used for detection of benzoic acid in food samples and was observed to have comparable results to the HPLC method, hence the developed test strip can be used as an alternative to HPLC in detecting benzoic acid in food products.

  3. Efficient Audio Power Amplification - Challenges

    Andersen, Michael Andreas E.

    2005-01-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where...

  4. Gene amplification during myogenic differentiation

    Fischer, Ulrike; Ludwig, Nicole; Raslan, Abdulrahman; Meier, Carola; Meese, Eckart

    2016-01-01

    Gene amplifications are mostly an attribute of tumor cells and drug resistant cells. Recently, we provided evidence for gene amplifications during differentiation of human and mouse neural progenitor cells. Here, we report gene amplifications in differentiating mouse myoblasts (C2C12 cells) covering a period of 7 days including pre-fusion, fusion and post-fusion stages. After differentiation induction we found an increase in copy numbers of CDK4 gene at day 3, of NUP133 at days 4 and 7, and of MYO18B at day 4. The amplification process was accompanied by gamma-H2AX foci that are indicative of double stand breaks. Amplifications during the differentiating process were also found in primary human myoblasts with the gene CDK4 and NUP133 amplified both in human and mouse myoblasts. Amplifications of NUP133 and CDK4 were also identified in vivo on mouse transversal cryosections at stage E11.5. In the course of myoblast differentiation, we found amplifications in cytoplasm indicative of removal of amplified sequences from the nucleus. The data provide further evidence that amplification is a fundamental mechanism contributing to the differentiation process in mammalians. PMID:26760505

  5. Efficient audio power amplification - challenges

    Andersen, Michael A.E.

    2005-07-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where extensive research and development are needed is covered. (au)

  6. Impedimetric test for rapid determination of performic acid (PFA biocidal activity toward Echerichia coli

    Małgorzata Lasik

    2013-12-01

    Full Text Available   Background. Performic acid has recently become available on a commercial scale for potential use in waste-water disinfection and can become an innovative biocide for various purposes in food processing. The aim of our study was: 1 to investigate the antimicrobial resistance of performic acid as high active and non toxic chemical disinfectant against Escherichi coli (hygiene indicator test  microorganism used in industrial micro- biology and 2 to evaluate the electrical impedance measurement method usefulness for fast and high precise test of antibacterial activity. Material and methods. Four types of antimicrobial disinfectants (commercial 35% hydrogen peroxide, 1% performic acid, 35% hydrogen peroxide and 15% formic acid were tested against Escherichia coli as hygiene indicator test microorganism. By evaluating the biocidal activity of selected disinfectants two methods were compared: electrical impedance measurement and classical serial dilution method with turbidity effect. Results.  It was stated that the performic acid expressed the highest antibacterial activity in comparison to other tested peroxide disinfectants: commercial 35% hydrogen peroxide solution and components required for performic acid production: 35% hydrogen peroxide solution with stabilizers and 15% formic acid solution with stabilizers. It was demonstrated that the proposed alternative microbiology method of electrical imped- ance measurement facilitates a rapidly and more precise analyses of the intensity of disinfectants inhibition effect. Conclusions. It can be postulated that both, the performic acid disinfectants as well as the impedimetric method can be a good advantage in the industrial microbiology.  

  7. PCR amplification on microarrays of gel immobilized oligonucleotides

    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.

  8. Evidence of high-elevation amplification versus Arctic amplification

    Qixiang Wang; Xiaohui Fan; Mengben Wang

    2016-01-01

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961–2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification b...

  9. Experimentally Testing the Hypothesis of a Limited Amino Acid Repertoire in Primitive Proteins

    Akanuma, S.; Nakajima, Y.; Yokobori, S.; Yamagishi, A.

    2013-11-01

    It has been argued that a fewer amino acids were used in primitive proteins and later the repertoire increased up to 20. To test this hypothesis experimentally, we restricted the amino acid usage of a reconstructed, ancestral protein to reduced sets.

  10. Development and performance test of a continuous source of nitrous acid (HONO)

    Ammann, M.; Roessler, E.; Kalberer, M.; Bruetsch, S.; Schwikowski, M.; Baltensperger, U.; Zellweger, C.; Gaeggeler, H.W. [Paul Scherrer Inst. (PSI), Villigen (Switzerland)

    1997-09-01

    Laboratory investigations involving nitrous acid (HONO) require a stable, continuous source of HONO at ppb levels. A flow type generation system based on the reaction of sodium nitrite with sulfuric acid has been developed. Performance and speciation of gaseous products were tested with denuder and chemiluminescence techniques. (author) 2 figs., 2 refs.

  11. An integrated disposable device for DNA extraction and helicase dependent amplification

    Mahalanabis, Madhumita; Do, Jaephil; ALMuayad, Hussam; Zhang, Jane Y.; Klapperich, Catherine M.

    2010-01-01

    Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a dis...

  12. Thermogravimetric and DSC testing of poly(lactic acid) nanocomposites

    Highlights: • The presence of nanoadditivities in PLA matrix improves thermal stability of PLA. • Shielding effect main reason for PLA thermal stability by nanofillers. • Thermal degradation suppression in UV-irradiated PLA by the removing of unstable compounds. - Abstract: Polymer nanocomposites based on poly(lactic acid), PLA, and two types of nanofillers: nanosilver and nanoclay were obtained by casting method. The thermal properties of PLA and nanocomposites have been studied by thermogravimetric analysis (TA) and differential scanning calorimetry (DSC). All samples have been UV-irradiated and the effect of photoprocess on their thermal stability has been estimated. It was found that nanoadditives and UV irradiation causes an increase of the activation energy of PLA thermal decomposition. DSC result supplies information on glass transition and crystallization/melting processes in PLA in the presence of nanosilver or nanoclay, also after exposure to UV

  13. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.

    Rodriguez-Manzano, Jesus; Karymov, Mikhail A; Begolo, Stefano; Selck, David A; Zhukov, Dmitriy V; Jue, Erik; Ismagilov, Rustem F

    2016-03-22

    Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests. PMID:26900709

  14. Post-Fragmentation Whole Genome Amplification-Based Method

    Benardini, James; LaDuc, Myron T.; Langmore, John

    2011-01-01

    This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at

  15. Next generation Chirped Pulse Amplification

    Nees, J.; Biswal, S.; Mourou, G. [Univ. Michigan, Center for Ultrafast Optical Science, Ann Arbor, MI (United States); Nishimura, Akihiko; Takuma, Hiroshi

    1998-03-01

    The limiting factors of Chirped Pulse Amplification (CPA) are discussed and experimental results of CPA in Yb:glass regenerative amplifier are given. Scaling of Yb:glass to the petawatt level is briefly discussed. (author)

  16. Modified performance test of vented lead acid batteries for stationary applications

    The concept of a modified performance test for vented lead acid batteries in stationary applications has been developed by the IEEE Battery Working Group. The modified performance test is defined as a test in the ''as found'' condition of the battery capacity and its ability to provide a high rate, short duration load (usually the highest rate of the duty cycle) that will confirm the battery's ability to meet the critical period of the load duty cycle, in addition to determining its percentage of rated capacity. This paper will begin by reviewing performance and service test requirements and concerns associated with both types of tests. The paper will then discuss the rationale for developing a modified performance test along with the benefits that can be derived from performing a modified performance test in lieu of a capacity test and/or a service test. The paper will conclude with an example on how to apply a modified performance test and test acceptance criteria

  17. Performance Degradation Tests of Phosphoric Acid Doped PBI Membrane Based High Temperature PEM Fuel Cells

    Zhou, Fan; Araya, Samuel Simon; Grigoras, Ionela;

    2014-01-01

    Degradation tests of two phosphoric acid (PA) doped PBI membrane based HT-PEM fuel cells were reported in this paper to investigate the effects of start/stop and the presence of methanol in the fuel to the performance degradation. Continuous tests with H2 and simulated reformate which was composed...

  18. Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

    Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, J Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, Maria Jesús

    2015-08-18

    Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas. PMID:26198403

  19. K Basin Sludge Conditioning Testing. Nitric Acid Dissolution Testing of K East Area Sludge Composite, Small- and Large-Scale Testing

    This report describes work performed by Pacific Northwest National Laboratory (PNNL) for Numatec Hanford Corporation (NHC) to support the development of the K Basin Sludge Treatment System. For this work, testing was performed to examine the dissolution behavior of a K East Basin floor and Weasel Pit sludge composite, referred to as K East area sludge composite, in nitric acid at the following concentrations: 2 M, 4 M, 6 M and 7.8 M. With the exception of one high solids loading test the nitric acid was added at 4X the stoichiometric requirement (assuming 100% of the sludge was uranium metal). The dissolution tests were conducted at boiling temperatures for 24 hours. Most of the tests were conducted with approximately2.5 g of sludge (dry basis). The high solids loading test was conducted with approximately7 g of sludge. A large-scale dissolution test was conducted with 26.5 g of sludge and 620 mL of 6 M nitric acid. The objectives of this test were to (1) generate a sufficient quantity of acid-insoluble residual solids for use in leaching studies, and (2) examine the dissolution behavior of the sludge composite at a larger scale

  20. Gold nanoparticle-based lateral flow biosensor for rapid visual detection of Leishmania-specific DNA amplification products.

    Toubanaki, Dimitra K; Athanasiou, Evita; Karagouni, Evdokia

    2016-08-01

    Leishmaniasis is a disease, caused by Leishmania parasites, which infect humans and animals, posing a major social and economic burden worldwide. The need for accurate and sensitive disease diagnosis led to the widespread adoption of PCR amplification. Detection of the amplification products (i.e. gel electrophoresis) require time-consuming protocols performed by trained personnel, with high cost. Aim of the present study was the simplification of PCR product detection, using a nucleic acid lateral flow, combined with functionalized gold nanoparticles. Amplification reactions targeting kinetoplastid DNA of Leishmania spp were performed on canine blood samples and a positive signal was formed as a red test zone. The visual detection was completed in 20min. Extensive optimization enabled the detection of 100fmol of target DNA. Clinical samples of infected dog blood were analyzed with high specificity. Overall, the proposed lateral flow biosensor can be considered an appealing alternative platform for Leishmania-specific amplification products detection with low cost and attractive simplicity. PMID:27255490

  1. Supplementary Testing Is Not Required in the cobas 4800 CT/NG Test for Neisseria gonorrhoeae Weak-Positive Urogenital Samples

    Bromhead, Collette; Liyanarachchy, Nadika; Mayes, Julia; Upton, Arlo; Balm, Michelle

    2014-01-01

    Weak-positive Neisseria gonorrhoeae nucleic acid amplification test results are difficult to interpret. We show that the frequency of unconfirmed N. gonorrhoeae results from the cobas 4800 test rises exponentially after 38.0 cycles, where the likelihood of an unconfirmed result exceeds 29%. Supplementary testing of such samples should be avoided; instead, treatment should be based on clinical pretest probability.

  2. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples. PMID:25822163

  3. Impedimetric test for rapid determination of performic acid (PFA) biocidal activity toward Echerichia coli

    Małgorzata Lasik; Renata Dobrucka; Piotr Konieczny

    2013-01-01

      Background. Performic acid has recently become available on a commercial scale for potential use in waste-water disinfection and can become an innovative biocide for various purposes in food processing. The aim of our study was: 1) to investigate the antimicrobial resistance of performic acid as high active and non toxic chemical disinfectant against Escherichi coli (hygiene indicator test  microorganism used in industrial micro- biology) and 2) to evaluate the electrical impedanc...

  4. Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.

    Cassol, S A; Poon, M.C.; Pal, R.; Naylor, M J; Culver-James, J; Bowen, T.J.; Russell, J A; Krawetz, S A; Pon, R T; Hoar, D I

    1989-01-01

    A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the a...

  5. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

    Ahmed Abd El Wahed

    Full Text Available Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF. Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR are the standard method for molecular detection of the dengue virus (DENV. Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA assays were developed to detect DENV1-4.Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4 to 241 (DENV1-3 RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal and in Bangkok (Thailand. In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31 and 100% (n=23, respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90 and 100%(n=41, respectively.During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

  6. Feedback Amplification of Neutrophil Function.

    Németh, Tamás; Mócsai, Attila

    2016-06-01

    As the first line of innate immune defense, neutrophils need to mount a rapid and robust antimicrobial response. Recent studies implicate various positive feedback amplification processes in achieving that goal. Feedback amplification ensures effective migration of neutrophils in shallow chemotactic gradients, multiple waves of neutrophil recruitment to the site of inflammation, and the augmentation of various effector functions of the cells. We review here such positive feedback loops including intracellular and autocrine processes, paracrine effects mediated by lipid (LTB4), chemokine, and cytokine mediators, and bidirectional interactions with the complement system and with other immune and non-immune cells. These amplification mechanisms are not only involved in antimicrobial immunity but also contribute to neutrophil-mediated tissue damage under pathological conditions. PMID:27157638

  7. Method for chemical amplification based on fluid partitioning in an immiscible liquid

    Anderson, Brian L.; Colston, Bill W.; Elkin, Christopher J.

    2015-06-02

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  8. Chemical amplification of magnetic field effects relevant to avian magnetoreception

    Kattnig, Daniel R.; Evans, Emrys W.; Déjean, Victoire; Dodson, Charlotte A.; Wallace, Mark I.; MacKenzie, Stuart R.; Timmel, Christiane R.; Hore, P. J.

    2016-04-01

    Magnetic fields as weak as the Earth's can change the yields of radical pair reactions even though the energies involved are orders of magnitude smaller than the thermal energy, kBT, at room temperature. Proposed as the source of the light-dependent magnetic compass in migratory birds, the radical pair mechanism is thought to operate in cryptochrome flavoproteins in the retina. Here we demonstrate that the primary magnetic field effect on flavin photoreactions can be amplified chemically by slow radical termination reactions under conditions of continuous photoexcitation. The nature and origin of the amplification are revealed by studies of the intermolecular flavin-tryptophan and flavin-ascorbic acid photocycles and the closely related intramolecular flavin-tryptophan radical pair in cryptochrome. Amplification factors of up to 5.6 were observed for magnetic fields weaker than 1 mT. Substantial chemical amplification could have a significant impact on the viability of a cryptochrome-based magnetic compass sensor.

  9. Testing for spatial clustering of amino acid replacements within protein tertiary structure

    Yu, Jiaye; Thorne, Jeffrey L

    2006-01-01

    Widely used models of protein evolution ignore protein structure. Therefore, these models do not predict spatial clustering of amino acid replacements with respect to tertiary structure. One formal and biologically implausible possibility is that there is no tendency for amino acid replacements to...... be spatially clustered during evolution. An alternative to this is that amino acid replacements are spatially clustered and this spatial clustering can be fully explained by a tendency for similar rates of amino acid replacement at sites that are nearby in protein tertiary structure. A third...... possibility is that the amount of clustering exceeds that which can be explained solely on the basis of independently evolving protein sites with spatially clustered replacement rates. We introduce two simple and not very parametric hypothesis tests that help distinguish these three possibilities. We then...

  10. Characterization of acidic and basic properties of heterogeneous catalysts by test reactions

    Alsawalha, Murad

    2005-01-01

    Classification of catalysts according to their active sites can be done by catalytic test reactions. In contrast to acid catalysed test reactions which are actually well understood there is a need to study base catalysed reactions in detail. Therefore reactions in gas phase like conversion of methyl butynol (MBOH) and isopropanol and in liquid phase like Knoevenagel condensation were investigated. It was found that the conversion of isopropanol yields propene as the only product over the cata...

  11. Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

    SA Faggion

    2013-01-01

    Full Text Available The rickettsial bacterium Ehrlichia canis is the etiological agent of canine monocytic ehrlichiosis, one of the most important canine tick-borne diseases in the world. In this study, a loop-mediated isothermal amplification (LAMP assay was developed for detection of E. canis DNA using LAMP primers targeting the groESL operon. Reactions were performed at 60°C for 60 min and the results were visualized by gel electrophoresis. Successful amplification was obtained using plasmid DNA containing a fragment of the groESL operon and DNA extracted from blood samples that tested positive for E. canis by real-time PCR. The specificity of amplification was confirmed by EcoRI restriction of internal sites in the LAMP primers and no cross-reactivity with blood samples positive for Babesia spp., another common tick-borne pathogen, was observed. The high cost of nucleic acid tests (NAT is one of the disadvantages for their large-scale use as routine diagnostic tests. The E. canis LAMP assay developed here is an interesting alternative to PCR since it does not require a thermocycler, thus reducing costs for the veterinary clinical laboratory.

  12. 21 CFR 862.1255 - 2,3-Diphosphoglyceric acid test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false 2,3-Diphosphoglyceric acid test system. 862.1255 Section 862.1255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  13. 21 CFR 862.1060 - Delta-aminolevulinic acid test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Delta-aminolevulinic acid test system. 862.1060 Section 862.1060 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  14. PYROLYTIC PRODUCTS FROM TRYPTOPHAN AND GLUTAMIC-ACID ARE POSITIVE IN THE MAMMALIAN SPOT-TEST

    Jensen, Niels Juul

    1983-01-01

    Pyrolysates of tryptophan (Trp-P-2) and glutamic acid (Glu-P-1) are known mutagens in in vitro short term mutagenicity tests, and have also shown carcinogenic effects in long term animal studies. The present study demonstrates that they also produce mutations in somatic cells. This result demonst...

  15. Development and testing of a bipolar lead-acid battery for hybrid electric vehicles

    Saakes, M.; Kluiters, E.; Schmal, D.; Mourad, S.; Have, P.T.J.H. ten

    1999-01-01

    An 80 V bipolar lead-acid battery was constructed and tested using hybrid electric vehicle (HEV) drive cycles. Drive cycles with a peak power of 6.7 kW, equal to 1/5 of the total power profile required for the HEV studied, were run successfully. Model calculations showed that the 80 V module constru

  16. Gas diffusion electrode setup for catalyst testing in concentrated phosphoric acid at elevated temperatures

    Wiberg, Gustav Karl Henrik; Fleige, Michael; Arenz, Matthias

    2015-01-01

    We present a detailed description of the construction and testing of an electrochemical cell setup allowing the investigation of a gas diffusion electrode containing carbon supported high surface area catalysts. The setup is designed for measurements in concentrated phosphoric acid at elevated...

  17. Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens

    We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in <20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics. (author)

  18. Gas amplification properties of GEM foils

    In the framework of the detector concept International Linear Detector for the future accelerator project International Linear Collider, in which electrons and positrons at c. m. energies of 500 GeV are brought to collision, a time projection chamber shall be applied as central track detector. By the application of such a chamber as track detector a three-dimensional reconstruction of the track points is possible. If a particle passes the gas volume within the chamber it ionizises single gas atoms and the arising electrons move after the amplification in the GEM arrangement to the anode, so that a two-dimensional projection of the particle track is possible. The third dimension is calculated from the drift time of the electrons. The advances of this readout system consist therein that a better position resolution than by a multiwire proportional chamber is reached and the back-drifting ions can be strongly suppressed. Aim of this thesis are studies for a GEM module, which shall be used in a large TPC prototype. Concerning different requirements it is valid to compare different GEMs in order to can meet an optimal choice. In a small prototype present at DESY measurements for the acquisition of GEM-describing parameters were performed. The taking into operation of the test TPC was part of this thesis. Tracks were generated by a radioactive source, by means of which the gas amplification was determined. With the measurement arrangement gas-amplifier foils of different kind were compared in view of their amplification properties and their energy resolution power and systematically studied. Five different GEM performances were studied in the test TPC. These foils differ in their geometrical classification parameters, the fabrication process, or the materials. The GEMs produced at CERN possess in comparison with GEMs of the Japanese firm SciEnergy and a GEM of the US-American firm Tech-Etch the best amplification and resolution properties. Furthermore a new GEM framing

  19. Genome position and gene amplification

    Jirsová, Pavla; Snijders, A.M.; Kwek, S.; Roydasgupta, R.; Fridlyand, J.; Tokuyasu, T.; Pinkel, D.; Albertson, D. G.

    2007-01-01

    Roč. 8, č. 6 (2007), r120. ISSN 1474-760X Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : gene amplification * array comparative genomic hybridization * oncogene Subject RIV: BO - Biophysics Impact factor: 6.589, year: 2007

  20. Strand Invasion Based Amplification (SIBA®: a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

    Mark J Hoser

    Full Text Available Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA. SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

  1. The neutralization of acidic coal mine lakes by additions of natural organic matter: a mesocosm test

    Cylindrical polyethylene enclosures 3 m in length and 1 m in diameter reaching from the surface to the bottom were constructed in an acid (pH=3.1) lake on a coal surface mine in southern Illinois. Wheat straw was added to the enclosures to test the effects of dissimilatory sulfate reduction on water chemistry. Added straw increased sulfide concentrations, raised pH to 6.5, reduced O2 and increased acid neutralizing capacity of the enclosed water columns when compared with a control enclosure and with the open lake. Generation of acid neutralizing capacity exceeded the standing stock of sulfide indicating that sulfide was removed either by precipitation of FeS or outgassing of H2S. The pH and acid neutralizing capacity within the enclosures eventually returned to the level of the surrounding lake because of water exchange around the enclosure walls. Our results show that additions of organic matter to acid surface mine lakes result in the generation of acid neutralizing capacity

  2. Double regenerative amplification of picosecond pulses

    Bai, Zhen-ao; Chen, Li-yuan; Bai, Zhen-xu; Chen, Meng; Li, Gang

    2012-04-01

    An double Nd:YAG regenerative amplification picosecond pulse laser is demonstrated under the semiconductor saturable absorption mirror(SESAM) mode-locking technology and regenerative amplification technology, using BBO crystal as PC electro-optic crystal. The laser obtained is 20.71ps pulse width at 10 KHz repetition rate, and the energy power is up to 4W which is much larger than the system without pre-amplification. This result will lay a foundation for the following amplification.

  3. Performace Test and Engine Emission on Acid Oleic Oxygenated as Additives Petrol

    Irfan Wahyudi; Abdul Amir Hassan Kadhum; Yusoff Ali

    2010-01-01

    The objective of this study is to investigate the effect of oxygenated oleic acid additives in standard petrol on the engine performance and the exhaust emissions. The 4-stroke 1.5 litre engine was used on the engine test bed coupled to eddy current electric dynamometer which is also connected to CADET V12 control system and exhaust gas analyser IMR2000/2000P. The oxygenated oleic acid additives used for this experiment is fixed at 0.2% by volume due to limited quantity available. Two types...

  4. Radiological bile acid absorption test 75SeHCAT in patients with diarrhoea of unknown cause

    Purpose: The purpose with this study was to investigate the usefulness of the 75SeHCAT test in patients suffering from 'functional' diarrhoea and to disclose earlier radiological investigations performed in the course of disease. Material and Methods: Twenty patients with chronic diarrhoea of unknown cause were investigated with the 75SeHCAT test. The cholestyramine test and an estimation of oro-caecal transit time (OCTT) were also performed in 13 and 10 patients respectively. Results: Ten patients had low 7-day retention (≤15%). Nine of these were tested with cholestyramine and 8 showed complete relief from the diarrhoea. The cholestyramine test had no effect on the 4 patients with normal bile acid retention. No correlation was found between the retention of 75SeHCAT and OCCT. A total of 80 inconclusive gastrointestinal barium examinations were performed during the course of disease. Conclusion: Pathological bile acid retention, studied with the radiological 75SeHCAT test, is common in patients with diarrhoea of unknown cause. The test should be performed earlier in the course of disease in order to prevent unnecessary radiological investigations and to reduce patient suffering and distress. (orig.)

  5. Gastric emptying of a solid meal starts during meal ingestion : Combined study using C-13-octanoic acid breath test and Doppler ultrasonography - Absence of a lag phase in C-13-octanoic acid breath test

    Minderhoud, IM; Mundt, MW; Roelofs, JMM; Samsom, M

    2004-01-01

    Scintigraphy and the C-13-octanoic acid breath test are both applied to assess gastric emptying. Using the C-13-octanoic acid breath test, excretion curves show C-13 excretion immediately after ingestion of a solid egg meal, in contrast with scintigraphy where gastric emptying is observed after a la

  6. Comprehensive human genome amplification using multiple displacement amplification

    Dean, Frank B.; Hosono, Seiyu; Fang, Linhua; Wu, Xiaohong; Faruqi, A. Fawad; Bray-Ward, Patricia; Zhenyu SUN; Zong, Qiuling; Du, Yuefen; Du, Jing; Driscoll, Mark; Song, Wanmin; Kingsmore, Stephen F.; Egholm, Michael; Lasken, Roger S.

    2002-01-01

    Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multi...

  7. TESTING OF 304L STAINLESS STEEL IN NITRIC ACID ENVIRONMENTS WITH FLUORIDES AND CHLORIDES

    Mickalonis, J.

    2010-10-04

    Impure radioactive material processed in nitric acid solutions resulted in the presence of chlorides in a dissolver fabricated from 304L stainless steel. An experimental program was conducted to study the effects of chloride in nitric acid/fluoride solutions on the corrosion of 304L stainless steel. The test variables included temperature (80, 95, and 110 C) and the concentrations of nitric acid (6, 12, and 14 M), fluoride (0.01, 0.1, and 0.2 M) and chloride (100, 350, 1000, and 2000 ppm). The impact of welding was also investigated. Results showed that the chloride concentration alone was not a dominant variable affecting the corrosion, but rather the interaction of chloride with fluoride significantly affected corrosion.

  8. Performace Test and Engine Emission on Acid Oleic Oxygenated as Additives Petrol

    Irfan Wahyudi

    2010-10-01

    Full Text Available The objective of this study is to investigate the effect of oxygenated oleic acid additives in standard petrol on the engine performance and the exhaust emissions. The 4-stroke 1.5 litre engine was used on the engine test bed coupled to eddy current electric dynamometer which is also connected to CADET V12 control system and exhaust gas analyser IMR2000/2000P. The oxygenated oleic acid additives used for this experiment is fixed at 0.2% by volume due to limited quantity available. Two types of test were carried out first test is with variable speed and no loads and the second test is at constant speed of 1,400 rpm with variable loads. The experimental results showed that the brake power increased by 2%, torque by 2%, brake thermal efficiency by about 7% and specific fuel consumption decreased by about 10%. The exhaust emissions analysed are carbon monoxide, (CO, carbon dioxide (CO2 and oxides of nitrogen (NOx. The result by comparing the oxygenated petrol with the standard petrol indicated that CO gas emission depend on the engine speed which decreased with increasing speed up to 1,600 rpm and increased after that speed on test without load. The CO also decreased by about 28% on the test with loads. The emission of CO2 increased by 2.7% for tests with load and by 8% for tests without loads. The NOx emission decreased by 9% for test without load and about 7% for test with load. This study indicates that engine performance is improved by adding 0.2% oxygenated additives to standard petrol.

  9. Skin testing of gallic acid-based hair dye in paraphenylenediamine/paratoluenediamine-reactive patients.

    Choi, Yunseok; Lee, Joon Ho; Kwon, Hyok Bu; An, Susun; Lee, Ai-Young

    2016-07-01

    Incidence of allergic contact dermatitis (ACD) to para-phenylenediamine (PPD)/paratoluenediamine (PTD) hair dyes is increasing. Hair dyes utilizing gallic acid (GA) may be a safe alternative. However, pretesting is recommended. We investigated the contact sensitivity to ingredients of a dye product; GA, monoethanolamine thioglycolate (MT), l-cystein and ferrous sulfate, and an appropriate pretest method in 31 patients reactive to PPD and/or PTD. An open test was performed with the test dye following the patch test. Subsequently, a use test was performed twice, with a 4-week interval. One subject showed a positive reaction to ferrous sulfate in the patch test. Another subject reacted to the first compound alone in the open test. Thirteen subjects manifesting cutaneous lesions from previous regular hair dyeing, showed reactions at the first use of the test dye; and six had reactions with reduced severity at the second test. GA and MT are safe for use in ACD patients reactive to PPD and/or PTD. For predicting contact allergy to hair dyes, the open test appeared to be a better pretest method than the patch test. PMID:26663148

  10. Regioselective synthesis of α-D-glucopyranosiduronic acid derivatives and biological test against bacterial Staphylococcus aureus and Salmonella agona

    CASSIDY; Seamas

    2010-01-01

    α-D-Glucopyranosiduronides were stereoselectively synthesized from D-glucopyranosiduronic acid via the intermediate 1,6-lactone. Fatty acid esters were regioselectively introduced at the 2-O-position of α-D-glucopyranosiduronides using dibutyltin dimethoxide as the stannylating agent. Antibacterial tests show that methyl 2-O-lauroyl-O-D-glucopyranosiduronic acid and azido 2-O-lauroyl-O-D-glucopyranosid-uronic acid are effective inhibitors against Staphylococcus aureus.

  11. 21 CFR 862.1055 - Newborn screening test system for amino acids, free carnitine, and acylcarnitines using tandem...

    2010-04-01

    ..., free carnitine, and acylcarnitines using tandem mass spectrometry. 862.1055 Section 862.1055 Food and... screening test system for amino acids, free carnitine, and acylcarnitines using tandem mass spectrometry. (a) Identification. A newborn screening test system for amino acids, free carnitine, and acylcarnitines using...

  12. 78 FR 58574 - Maintenance, Testing, and Replacement of Vented Lead-Acid Storage Batteries for Nuclear Power Plants

    2013-09-24

    ... COMMISSION Maintenance, Testing, and Replacement of Vented Lead-Acid Storage Batteries for Nuclear Power..., Testing, and Replacement of Vented Lead-Acid Storage Batteries for Nuclear Power Plants.'' The guide... nuclear power plants. ADDRESSES: Please refer to Docket ID NRC-2013-0048 when contacting the NRC about...

  13. In vitro testing of thiolated poly(aspartic acid) from ophthalmic formulation aspects.

    Budai-Szű Cs, Mária; Horvát, Gabriella; Gyarmati, Benjámin; Szilágyi, Barnabás Áron; Szilágyi, András; Csihi, Tímea; Berkó, Szilvia; Szabó-Révész, Piroska; Mori, Michela; Sandri, Giuseppina; Bonferoni, Maria Cristina; Caramella, Carla; Csányi, Erzsébet

    2016-08-01

    Ocular drug delivery formulations must meet anatomical, biopharmaceutical, patient-driven and regulatory requirements. Mucoadhesive polymers can serve as a better alternative to currently available ophthalmic formulations by providing improved bioavailability. If all requirements are addressed, a polymeric formulation resembling the tear film of the eye might be the best solution. The optimum formulation must not have high osmotic activity, should provide appropriate surface tension, pH and refractive index, must be non-toxic and should be transparent and mucoadhesive. We would like to highlight the importance of in vitro polymer testing from a pharmaceutical aspect. We, therefore, carried out physical-chemical investigations to verify the suitability of certain systems for ophthalmic formulations. In this work, in situ gelling, mucoadhesive thiolated poly(aspartic acid)s were tested from ophthalmic formulation aspects. The results of preformulation measurements indicate that these polymers can be used as potential carriers in ophthalmic drug delivery. PMID:26556306

  14. DEPOSITION TANK CORROSION TESTING FOR ENHANCED CHEMICAL CLEANING POST OXALIC ACID DESTRUCTION

    Mickalonis, J.

    2011-08-29

    An Enhanced Chemical Cleaning (ECC) process is being developed to aid in the high level waste tank closure at the Savannah River Site. The ECC process uses an advanced oxidation process (AOP) to destroy the oxalic acid that is used to remove residual sludge from a waste tank prior to closure. The AOP process treats the dissolved sludge with ozone to decompose the oxalic acid through reactions with hydroxyl radicals. The effluent from this oxalic acid decomposition is to be sent to a Type III waste tank and may be corrosive to these tanks. As part of the hazardous simulant testing that was conducted at the ECC vendor location, corrosion testing was conducted to determine the general corrosion rate for the deposition tank and to assess the susceptibility to localized corrosion, especially pitting. Both of these factors impact the calculation of hydrogen gas generation and the structural integrity of the tanks, which are considered safety class functions. The testing consisted of immersion and electrochemical testing of A537 carbon steel, the material of construction of Type III tanks, and 304L stainless steel, the material of construction for transfer piping. Tests were conducted in solutions removed from the destruction loop of the prototype ECC set up. Hazardous simulants, which were manufactured at SRNL, were used as representative sludges for F-area and H-area waste tanks. Oxalic acid concentrations of 1 and 2.5% were used to dissolve the sludge as a feed to the ECC process. Test solutions included the uninhibited effluent, as well as the effluent treated for corrosion control. The corrosion control options included mixing with an inhibited supernate and the addition of hydroxide. Evaporation of the uninhibited effluent was also tested since it may have a positive impact on reducing corrosion. All corrosion testing was conducted at 50 C. The uninhibited effluent was found to increase the corrosion rate by an order of magnitude from less than 1 mil per year (mpy

  15. Lead-acid batteries in micro-hybrid applications. Part II. Test proposal

    Schaeck, S.; Stoermer, A.O. [BMW Group, 80788 Muenchen (Germany); Albers, J. [Johnson Controls Power Solutions EMEA, 30419 Hannover (Germany); Weirather-Koestner, D. [ZSW Ulm, 89081 Ulm (Germany); Kabza, H. [Universitaet Ulm, Institut fuer Energiewandlung und -speicherung, 89081 Ulm (Germany)

    2011-02-01

    In the first part of this work selected key parameters for applying lead-acid (LA) batteries in micro-hybrid power systems (MHPS) were investigated. Main results are integrated in an accelerated, comprehensive test proposal presented here. The test proposal aims at a realistic representation of the pSoC operation regime, which is described in Refs. The test is designed to be sensitive with respect to dynamic charge acceptance (DCA) at partially discharged state (critical for regenerative braking) and the internal resistance at high-rate discharge (critical for idling stop applications). First results are presented for up-to-date valve-regulated LA batteries with absorbent glass mat (AGM) separators. The batteries are close to the limits of the first proposal of pass/fail-criteria. Also flooded batteries were tested; the first out of ten units failed already. (author)

  16. Lead-acid batteries in micro-hybrid applications. Part II. Test proposal

    Schaeck, S.; Stoermer, A. O.; Albers, J.; Weirather-Koestner, D.; Kabza, H.

    In the first part of this work [1] selected key parameters for applying lead-acid (LA) batteries in micro-hybrid power systems (MHPS) were investigated. Main results are integrated in an accelerated, comprehensive test proposal presented here. The test proposal aims at a realistic representation of the pSoC operation regime, which is described in Refs. [1,6]. The test is designed to be sensitive with respect to dynamic charge acceptance (DCA) at partially discharged state (critical for regenerative braking) and the internal resistance at high-rate discharge (critical for idling stop applications). First results are presented for up-to-date valve-regulated LA batteries with absorbent glass mat (AGM) separators. The batteries are close to the limits of the first proposal of pass/fail-criteria. Also flooded batteries were tested; the first out of ten units failed already.

  17. 14C octanoic acid breath tests correlate with scintigraphy in the critically ill

    Full text: Gastric stasis is common in the critically ill and reduces tolerance to nasogastric feeding. Scintigraphic measurement of gastric emptying is rarely used in the critically ill for practical reasons. Breath testing would be a more convenient way of determining gastric emptying. The purpose of this study was to validate 14C octanoic acid breath tests against scintigraphy in ventilated patients. 21 unselected mechanically ventilated patients receiving enteral nutrition via a nasogastric tube (14M, mean age 58yrs) were fed a dual radiolabelled nutrient meal consisting of 100ml Ensure, 74KBq 14C octanoic acid and 20MBq 99Tc sulphur colloid. 12 normal volunteers (9M mean age 38yrs) were also studied to establish normal ranges. Dynamic scintigraphic images were collected with subjects in 20 deg LAO position over 4 hours utilising a GE Starcam 300M mobile gamma camera. Breath samples were collected using the RAH 'Rapid-14' collection methodology at baseline and every 10 -15 minutes for 4 hours. Gastric emptying parameters were calculated and correlated. Normal breath test mean t- = 117min (SD=46). Delayed gastric emptying was seen in some of the critically ill patients, mean t- - 143 mm (SD=86). Strongest correlations were between normals scintigraphic t- (t-s) and breath test t- (t-B), r=0.96 (p-B, r=0.75 (p-s and t-B, n=16, r=0.73 (p14C octanoic acid breath tests in normal subjects and the critically ill. This may be a useful alternative for measurement of gastric emptying in the critically ill. Copyright (2002) The Australian and New Zealand Society of Nuclear Medicine Inc

  18. Design and performance testing of a DNA extraction assay for sensitive and reliable quantification of acetic acid bacteria directly in red wine using real time PCR

    Cédric eLONGIN

    2016-06-01

    Full Text Available 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 mL 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.

  19. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  20. Loop-mediated isothermal amplification for detection of porcine circovirus type 2

    Zhou Shun; Han Si; Shi Jianli; Wu Jiaqiang; Yuan Xiaoyuan; Cong Xiaoyan; Xu Shaojian; Wu Xiaoyan; Li Jun; Wang Jinbao

    2011-01-01

    Abstract Background Porcine circovirus type 2 (PCV2) is the primary causative agent of the emerging swine disease known as postweaning multisystemic wasting syndrome (PMWS). Nowadays, polymerase chain reaction (PCR) is still the most widespread technique in pathogen detection. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method developed in 2000, will possibly replace PCR in the field of detection. To establish a LAMP method for rapid detection of PCV2, tw...

  1. Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K.; Klapperich, Catherine M.

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the...

  2. K Basin Sludge Conditioning Process Testing Project. Results from Test 4, ''Acid Digestion of Mixed-Bed Ion Exchange Resin''

    Approximately 73 m3 of heterogeneous solid material, ''sludge,'' (upper bound estimate, Packer 1997) have accumulated at the bottom of the K Basins in the 100 K Area of the Hanford Site. This sludge is a mixture of spent fuel element corrosion products, ion exchange materials (organic and inorganic), graphite-based gasket materials, iron and aluminum metal corrosion products, sand, and debris (Makenas et al. 1996, 1997). In addition, small amounts of polychlorinated biphenyls (PCBs) have been found. Ultimately, it is planned to transfer the K Basins sludge to the Hanford double shell tanks (DSTs). The Hanford Spent Nuclear Fuel (HSNF) project has conducted a number of evaluations to examine technology and processing alternatives to pretreat K Basin sludge to meet storage and disposal requirements. From these evaluations, chemical pretreatment has been selected to address criticality issues, reactivity, and the destruction or removal of PCBs before the K Basin sludge can be transferred to the DSTs. Chemical pretreatment, referred to as the K Basin sludge conditioning process, includes nitric acid dissolution of the sludge (with removal of acid insoluble solids), neutrons absorber addition, neutralization, and reprecipitation. Laboratory testing is being conducted by the Pacific Northwest National Laboratory (PNNL) to provide data necessary to develop the sludge conditioning process

  3. Tipepidine enhances the antinociceptive-like action of carbamazepine in the acetic acid writhing test.

    Kawaura, Kazuaki; Miki, Risa; Urashima, Yuri; Honda, Sokichi; Shehata, Ahmed M; Soeda, Fumio; Shirasaki, Tetsuya; Takahama, Kazuo

    2011-01-25

    Several antidepressants have been used to treat severe pain in clinics. Recently, we reported that the centrally acting non-narcotic antitussive (cough suppressant drug), tipepidine produces an antidepressant-like effect in the forced swimming test, although the mechanism of action appears to be quite different from that of known antidepressants. In the present study, we investigated whether a combination of tipepidine and carbamazepine acts synergistically to induce an antinociceptive effect in the acetic acid-induced writhing test in mice. Prior to studying the combination of tipepidine and carbamazepine, the analgesic action of tipepidine alone was also examined in mice. Tipepidine at 5-40mg/kg i.p. significantly reduced the number of writhes induced by acetic acid in mice. Carbamazepine at 20mg/kg i.p. also significantly reduced the writhing reaction. Furthermore, co-administration of carbamazepine (5 and 10mg/kg, i.p.) and tipepidine (2.5mg/kg i.p.) significantly decreased the number of writhes induced by acetic acid. This finding suggests that a combination of carbamazepine and tipepidine may be a new strategy for the treatment of neuropathic pain such as what occurs in trigeminal neuralgia, because the use of carbamazepine is often limited by its adverse effects and by reduction of its analgesic efficacy by microsomal enzyme induction. PMID:21114989

  4. Development of a loop-mediated isothermal amplification assay for rapid detection of Yersinia enterocolitica via targeting a conserved locus

    Reza Ranjbar

    2015-11-01

    Full Text Available Background and Objectives: Loop-mediated isothermal amplification is a novel nucleic acid amplification assay providing as a simple diagnostic tool for rapid identification of microbial diseases in developing countries. In this study, a LAMP assay was established for Yersinia enterocolitica, a leading cause of acute enterocolitis in young children.Materials and Methods: LAMP assay was established with four primers targeting a specific locus for the detection of Y. enterocolitica. The assay was conducted at 65°C in thermo block for 90min. The sensitivity of LAMP was evaluated in com- parison to conventional PCR using pTZ57R containing the target locus. Finally, specificity was assessed using DNA from common enteropathogenic bacteria.Results: Results showed that the sensitivity of LAMP assay was 44-copy number, which was 10-fold higher than that ofPCR. No cross-reactivity was observed when testing against other enteropathogenic pathogens.Conclusion: This study showed that LAMP assay is an alternative molecular diagnostic tool for infections with Y. enteroco- litica. In addition, this method may be useful in diagnosis at field or in laboratories without PCR machine.Keywords: Yersinia enterocolitica; Loop-mediated isothermal amplification (LAMP, specific locus 

  5. Ultrasmall volume molecular isothermal amplification in microfluidic chip with advanced surface processing

    In this paper, we developed a metal micro-fluidic chip with advanced surface processing for ultra-small volume molecular isothermal amplification. This method takes advantages of the nucleic acid amplification with good stability and consistency, high sensitivity about 31 genomic DNA copies and bacteria specific gene identification. Based on the advanced surface processing, the bioreaction assays of nucleic acid amplification was dropped about 392nl in volume. A high numerical aperture confocal optical detection system was advanced to sensitively monitor the DNA amplification with low noise and high power collecting fluorescence near to the optical diffraction limit. A speedy nucleic acid isothermal amplification was performed in the ultra-small volume microfluidic chip, where the time at the inflexions of second derivative to DNA exponential amplified curves was brought forward and the sensitivity was improved about 65 folds to that of in current 25μl Ep-tube amplified reaction, which indicates a promising clinic molecular diagnostics in the droplet amplification.

  6. Beyond the diffraction limit via optical amplification

    Kellerer, Aglae N

    2016-01-01

    In a previous article we suggested a method to overcome the diffraction limit behind a telescope. We refer to theory and recent numerical simulations, and test whether it is indeed possible to use photon amplification to enhance the angular resolution of a telescope or a microscope beyond the diffraction limit. An essential addition is the proposal to select events with above-average ratio of stimulated to spontaneous photons. We find that the diffraction limit of a telescope is surpassed by a factor ten for an amplifier gain of 200, if the analysis is restricted to a tenth of the incoming astronomical photons. A gain of 70 is sufficient with a hundredth of the photons.

  7. Integrated Amplification Microarrays for Infectious Disease Diagnostics

    Darrell P. Chandler

    2012-11-01

    Full Text Available This overview describes microarray-based tests that combine solution-phase amplification chemistry and microarray hybridization within a single microfluidic chamber. The integrated biochemical approach improves microarray workflow for diagnostic applications by reducing the number of steps and minimizing the potential for sample or amplicon cross-contamination. Examples described herein illustrate a basic, integrated approach for DNA and RNA genomes, and a simple consumable architecture for incorporating wash steps while retaining an entirely closed system. It is anticipated that integrated microarray biochemistry will provide an opportunity to significantly reduce the complexity and cost of microarray consumables, equipment, and workflow, which in turn will enable a broader spectrum of users to exploit the intrinsic multiplexing power of microarrays for infectious disease diagnostics.

  8. Telomerase Repeated Amplification Protocol (TRAP)

    Mender, Ilgen; Shay, Jerry W.

    2016-01-01

    Telomeres are found at the end of eukaryotic linear chromosomes, and proteins that bind to telomeres protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions (Griffith et al., 1999). However, due to the end replication problem and other factors such as oxidative damage, the limited life span of cultured cells (Hayflick limit) results in progressive shortening of these protective structures (Hayflick and Moorhead, 1961; Olovnikov, 1973). The ribonucleoprotein enzyme complex telomerase-consisting of a protein catalytic component hTERT and a functional RNA component hTR or hTERC- counteracts telomere shortening by adding telomeric repeats to the end of chromosomes in ~90% of primary human tumors and in some transiently proliferating stem-like cells (Shay and Wright, 1996; Shay and Wright, 2001). This results in continuous proliferation of cells which is a hallmark of cancer. Therefore, telomere biology has a central role in aging, cancer progression/metastasis as well as targeted cancer therapies. There are commonly used methods in telomere biology such as Telomere Restriction Fragment (TRF) (Mender and Shay, 2015b), Telomere Repeat Amplification Protocol (TRAP) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this detailed protocol we describe Telomere Repeat Amplification Protocol (TRAP). The TRAP assay is a popular method to determine telomerase activity in mammalian cells and tissue samples (Kim et al., 1994). The TRAP assay includes three steps: extension, amplification, and detection of telomerase products. In the extension step, telomeric repeats are added to the telomerase substrate (which is actually a non telomeric oligonucleotide, TS) by telomerase. In the amplification step, the extension products are amplified by the polymerase chain reaction (PCR) using specific primers (TS upstream primer and ACX downstream primer) and in the detection step, the presence or absence of telomerase is

  9. Resonant primordial gravitational waves amplification

    Chunshan Lin

    2016-01-01

    Full Text Available We propose a mechanism to evade the Lyth bound in models of inflation. We minimally extend the conventional single-field inflation model in general relativity (GR to a theory with non-vanishing graviton mass in the very early universe. The modification primarily affects the tensor perturbation, while the scalar and vector perturbations are the same as the ones in GR with a single scalar field at least at the level of linear perturbation theory. During the reheating stage, the graviton mass oscillates coherently and leads to resonant amplification of the primordial tensor perturbation. After reheating the graviton mass vanishes and we recover GR.

  10. Rapid Diagnosis of Extrapulmonary Tuberculosis by Ligase Chain Reaction Amplification

    Gamboa, Fredy; Dominguez, José; Padilla, Eduardo; Manterola, José M.; Gazapo, Elena; Lonca, Joan; Matas, Lurdes; Hernandez, Agueda; Cardona, Pere Joan; Ausina, Vicente

    1998-01-01

    A rapid amplification-based test for the diagnosis of extrapulmonary tuberculosis, the LCx Mycobacterium tuberculosis Assay from Abbott Laboratories, was evaluated. Results from the LCx M. tuberculosis Assay were compared with those from culture and the final clinical diagnosis for each patient. A total of 526 nonrespiratory specimens from 492 patients were tested. The specimens included urine; feces; lymph node exudates; pleural, cerebrospinal, articular, and ascitic fluids; tissue biopsies;...

  11. Dynamics and Control of DNA Sequence Amplification

    Marimuthu, Karthikeyan

    2014-01-01

    DNA amplification is the process of replication of a specified DNA sequence \\emph{in vitro} through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction (PCR) as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal tempe...

  12. Multiple displacement amplification as an adjunct to PCR-based detection of Staphylococcus aureus in synovial fluid

    Johnson Sandra

    2010-10-01

    Full Text Available Abstract Background Detection of bacterial nucleic acids in synovial fluid following total joint arthroplasty with suspected infection can be difficult; among other technical challenges, inhibitors in the specimens require extensive sample preparation and can diminish assay sensitivity even using polymerase chain reaction (PCR-based methods. To address this problem a simple protocol for prior use of multiple displacement amplification (MDA as an adjunct to PCR was established and tested on both purified S. aureus DNA as well as on clinical samples known to contain S. aureus nucleic acids. Findings A single round of MDA on purified nucleic acids resulted in a > 300 thousand-fold increase in template DNA on subsequent quantitative PCR (qPCR analysis. MDA use on clinical samples resulted in at least a 100-fold increase in sensitivity on subsequent qPCR and required no sample preparation other than a simple alkali/heat lysis step. Mixed samples of S. aureus DNA with a 103 - 104-fold excess of human genomic DNA still allowed for MDA amplification of the minor bacterial component to the threshold of detectability. Conclusion MDA is a promising technique that may serve to significantly enhance the sensitivity of molecular assays in cases of suspected joint infection while simultaneously reducing the specimen handling required.

  13. Modelling and Managing SSD Write-amplification

    Dayan, Niv; Bouganim, Luc; Bonnet, Philippe

    2015-01-01

    How stable is the performance of your flash-based Solid State Drives (SSDs)? This question is central for database designers and administrators, cloud service providers, and SSD constructors. The answer depends on write-amplification, i.e., garbage collection overhead. More specifically, the answer depends on how write-amplification evolves in time. How then can one model and manage write-amplification, especially when application workloads change? This is the focus of this paper. Managing wr...

  14. Validation Testing of the Nitric Acid Dissolution Step Within the K Basin Sludge Pretreatment Process

    AJ Schmidt; CH Delegard; KL Silvers; PR Bredt; CD Carlson; EW Hoppe; JC Hayes; DE Rinehart; SR Gano; BM Thornton

    1999-03-24

    The work described in this report involved comprehensive bench-scale testing of nitric acid (HNO{sub 3}) dissolution of actual sludge materials from the Hanford K East (KE) Basin to confirm the baseline chemical pretreatment process. In addition, process monitoring and material balance information was collected to support the development and refinement of process flow diagrams. The testing was performed by Pacific Northwest National Laboratory (PNNL)for the US Department of Energy's Office of Spent Fuel Stabilization (EM-67) and Numatec Hanford Corporation (NHC) to assist in the development of the K Basin Sludge Pretreatment Process. The baseline chemical pretreatment process for K Basin sludge is nitric acid dissolution of all particulate material passing a 1/4-in. screen. The acid-insoluble fraction (residual solids) will be stabilized (possibly by chemical leaching/rinsing and grouting), packaged, and transferred to the Hanford Environmental Restoration Disposal Facility (ERDF). The liquid fraction is to be diluted with depleted uranium for uranium criticality safety and iron nitrate for plutonium criticality safety, and neutralized with sodium hydroxide. The liquid fraction and associated precipitates are to be stored in the Hanford Tank Waste Remediation Systems (TWRS) pending vitrification. It is expected that most of the polychlorinated biphenyls (PCBs), associated with some K Basin sludges, will remain with the residual solids for ultimate disposal to ERDF. Filtration and precipitation during the neutralization step will further remove trace quantities of PCBs within the liquid fraction. The purpose of the work discussed in this report was to examine the dissolution behavior of actual KE Basin sludge materials at baseline flowsheet conditions and validate the.dissolution process step through bench-scale testing. The progress of the dissolution was evaluated by measuring the solution electrical conductivity and concentrations of key species in the

  15. Validation Testing of the Nitric Acid Dissolution Step Within the K Basin Sludge Pretreatment Process

    The work described in this report involved comprehensive bench-scale testing of nitric acid (HNO3) dissolution of actual sludge materials from the Hanford K East (KE) Basin to confirm the baseline chemical pretreatment process. In addition, process monitoring and material balance information was collected to support the development and refinement of process flow diagrams. The testing was performed by Pacific Northwest National Laboratory (PNNL)for the US Department of Energy's Office of Spent Fuel Stabilization (EM-67) and Numatec Hanford Corporation (NHC) to assist in the development of the K Basin Sludge Pretreatment Process. The baseline chemical pretreatment process for K Basin sludge is nitric acid dissolution of all particulate material passing a 1/4-in. screen. The acid-insoluble fraction (residual solids) will be stabilized (possibly by chemical leaching/rinsing and grouting), packaged, and transferred to the Hanford Environmental Restoration Disposal Facility (ERDF). The liquid fraction is to be diluted with depleted uranium for uranium criticality safety and iron nitrate for plutonium criticality safety, and neutralized with sodium hydroxide. The liquid fraction and associated precipitates are to be stored in the Hanford Tank Waste Remediation Systems (TWRS) pending vitrification. It is expected that most of the polychlorinated biphenyls (PCBs), associated with some K Basin sludges, will remain with the residual solids for ultimate disposal to ERDF. Filtration and precipitation during the neutralization step will further remove trace quantities of PCBs within the liquid fraction. The purpose of the work discussed in this report was to examine the dissolution behavior of actual KE Basin sludge materials at baseline flowsheet conditions and validate the.dissolution process step through bench-scale testing. The progress of the dissolution was evaluated by measuring the solution electrical conductivity and concentrations of key species in the dissolver

  16. Dynamics and control of DNA sequence amplification

    DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions

  17. Dynamics and control of DNA sequence amplification

    Marimuthu, Karthikeyan [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Chakrabarti, Raj, E-mail: raj@pmc-group.com, E-mail: rajc@andrew.cmu.edu [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Division of Fundamental Research, PMC Advanced Technology, Mount Laurel, New Jersey 08054 (United States)

    2014-10-28

    DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.

  18. Risk Perception and Social Amplification

    This paper seeks to consider social amplification as it applies to risk perception. Perceptions of the magnitude of a risk are conditioned by issues such as the degree of uncertainty in probability and consequences, the nature of the consequences and the relative weightings placed on probability and consequences. Risk perceptions are also influenced by factors such as confidence in the operator of an industrial process, trust in the regulator and the perceived fairness of regulatory decision-making. Different people may hold different views about these issues and there may also be difficulties in communication. The paper identifies and discusses self-reinforcing mechanisms, which will be labelled 'lock-in' here. They appear to apply in many situations where social amplification is observed. Historically, the term 'lock-in' has been applied mainly in the technological context but, in this paper, four types of lock-in are identified, namely scientific/technological, economic, social and institutional lock-in. One type of lock-in tends to lead to the next and all are buttressed by people's general acceptance of the familiar, fear of the unknown and resistance to change. The regulator seeks to make decisions which achieve the common good rather than supporting or perpetuating any set of vested interests. In this regard the locked-in positions of stakeholders, whether organisations, interest groups, or individual members of the public, are obstacles and challenges. Existing methods of consultation are unsatisfactory in terms of achieving a proper and productive level of dialogue with stakeholders

  19. CORROSION TESTING OF CARBON STEEL IN OXALIC ACID CHEMICAL CLEANING SOLUTIONS

    Wiersma, B.; Mickalonis, J.; Subramanian, K.; Ketusky, E.

    2011-10-14

    Radioactive liquid waste has been stored in underground carbon steel tanks for nearly 60 years at the Savannah River Site. The site is currently in the process of removing the waste from these tanks in order to place it into vitrified, stable state for longer term storage. The last stage in the removal sequence is a chemical cleaning step that breaks up and dissolves metal oxide solids that cannot be easily pumped out of the tank. Oxalic acid has been selected for this purpose because it is an effective chelating agent for the solids and is not as corrosive as other acids. Electrochemical and immersion studies were conducted to investigate the corrosion behavior of carbon steel in simulated chemical cleaning environments. The effects of temperature, agitation, and the presence of sludge solids in the oxalic acid on the corrosion rate and the likelihood of hydrogen evolution were determined. The testing showed that the corrosion rates decreased significantly in the presence of the sludge solids. Corrosion rates increased with agitation, however, the changes were less noticeable.

  20. Idling-stop vehicle road tests of advanced valve-regulated lead-acid (VRLA) battery

    Sawai, Ken; Ohmae, Takao; Suwaki, Hironori; Shiomi, Masaaki; Osumi, Shigeharu

    The results of road tests on valve-regulated lead-acid (VRLA) batteries in an idling-stop (stop and go) vehicle are reported. Idling-stop systems are simple systems to improve fuel economy of automobiles. They are expected to spread widely from an environmental perspective. Performances of a conventional flooded battery, a conventional VRLA battery, and an improved VRLA battery were compared in road tests with an idling-stop vehicle. It was found that the improved VRLA battery was suited to idling-stop applications because it had a smaller capacity loss than the conventional flooded battery during partial-state-of-charge (PSoC) operation. The positive grid was corroded in layers, unlike the usual grain boundary corrosion of SLI battery grid. It is because the corrosion proceeded mainly under PSoC conditions. The corrosion rate could be controlled by potential control of positive plates.

  1. Idling-stop vehicle road tests of advanced valve-regulated lead-acid (VRLA) battery

    Sawai, Ken; Ohmae, Takao; Suwaki, Hironori; Shiomi, Masaaki; Osumi, Shigeharu [Technical Development Division, Automotive Battery Business Unit, GS Yuasa Power Supply Ltd., Nishinosho, Kisshoin, Minami-ku, Kyoto (Japan)

    2007-11-22

    The results of road tests on valve-regulated lead-acid (VRLA) batteries in an idling-stop (stop and go) vehicle are reported. Idling-stop systems are simple systems to improve fuel economy of automobiles. They are expected to spread widely from an environmental perspective. Performances of a conventional flooded battery, a conventional VRLA battery, and an improved VRLA battery were compared in road tests with an idling-stop vehicle. It was found that the improved VRLA battery was suited to idling-stop applications because it had a smaller capacity loss than the conventional flooded battery during partial-state-of-charge (PSoC) operation. The positive grid was corroded in layers, unlike the usual grain boundary corrosion of SLI battery grid. It is because the corrosion proceeded mainly under PSoC conditions. The corrosion rate could be controlled by potential control of positive plates. (author)

  2. Small Sample Whole-Genome Amplification

    Hara, C A; Nguyen, C P; Wheeler, E K; Sorensen, K J; Arroyo, E S; Vrankovich, G P; Christian, A T

    2005-09-20

    Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.

  3. Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR.

    Zwadyk, P.; Down, J. A.; Myers, N; Dey, M. S.

    1994-01-01

    Two criteria must be met before mycobacterial specimens can be tested by DNA amplification methods: (i) the sample must be rendered noninfectious, and (ii) the organisms must be lysed to free the DNA. Previous publications reporting DNA amplification of mycobacteria have concentrated on lysis and amplification procedures and have not addressed the issue of sample safety. We have shown that heating of samples below 100 degrees C may not consistently kill mycobacteria; however, heating at 100 d...

  4. Comparison of the boronic acid disk potentiation test and cefepime-clavulanic acid method for the detection of ESBL among AmpC-producing Enterobacteriaceae

    R M Shoorashetty

    2011-01-01

    Full Text Available Purpose: Extended spectrum β-lactamase (ESBL and AmpC β-lactamase are important mechanisms of betalactam resistance among Enterobacteriaceae . The ESBL confirmation test described by Clinical Laboratory Standards Institute (CLSI is in routine use. This method fails to detect ESBL in the presence of AmpC. Therefore, we compared two different ESBL detection methods against the CLSI confirmatory test. Materials and Methods: A total 200 consecutive clinical isolates of Enterobacteriaceae from various clinical samples were tested for ESBL production using (i CLSI described phenotypic confirmatory test (PCT, (ii boronic acid disk potentiation test and (iii cefepime-CA disk potentiation method. AmpC confirmation was done by a modified three-dimensional test. Results: Among total 200 Enterobacteriaceae isolates, 82 were only ESBL producers, 12 were only AmpC producers, 55 were combined ESBL and AmpC producers, 14 were inducible AmpC producers and 37 isolates did not harboured any enzymes. The CLSI described PCT detected ESBL-producing organisms correctly but failed to detect 36.3% of ESBLs among combined enzyme producers. The boronic acid disk potentiation test reliably detected all ESBL, AmpC, and combined enzyme producers correctly. The cefepime-CA method detected all ESBLs correctly but another method of AmpC detection has to be adopted. Conclusion: The use of boronic acid in disk diffusion testing along with the CLSI described PCT enhances ESBL detection in the presence of AmpC betalactamases.

  5. Approaches towards molecular amplification for sensing.

    Goggins, Sean; Frost, Christopher G

    2016-06-01

    Diagnostic assays that rely on molecular interactions have come a long way; from initial reversible detection systems towards irreversible reaction indicator-based methods. More recently, the emergence of innovative molecular amplification methodologies has revolutionised sensing, allowing diagnostic assays to achieve ultra-low limits of detection. There have been a significant number of molecular amplification approaches developed over recent years to accommodate the wide variety of analytes that require sensitive detection. To celebrate this achievement, this comprehensive critical review has been compiled to give a broad overview of the many different approaches used to attain amplification in sensing with an aim to inspire the next generation of diagnostic assays looking to achieve the ultimate detection limit. This review has been created with the focus on how each conceptually unique molecular amplification methodology achieves amplification, not just its sensitivity, while highlighting any key processes. Excluded are any references that were not found to contain an obvious molecular amplifier or amplification component, or that did not use an appropriate signal readout that could be incorporated into a sensing application. Additionally, methodologies where amplification is achieved through advances in instrumentation are also excluded. Depending upon the type of approach employed, amplification strategies are divided into four categories: target, label, signal or receptor amplification. More recent, more complex protocols combine a number of approaches and are therefore categorised by which amplification component described within was considered as the biggest advancement. The advantages and disadvantages of each methodology are discussed along with any limits of detection, if stated in the original article. Any subsequent use of the methodology within sensing or any other application is also mentioned to draw attention to its practicality. The importance of

  6. Corrosion Testing of Carbon Steel in Oxalic Acid that Contains Dissolved Iron

    Wiersma, Bruce J.; Mickalonis, John I.; Subramanian, Karthik H.

    2012-10-11

    Radioactive liquid waste has been stored in underground carbon steel tanks for nearly 60 years at the Savannah River Site. The site is currently in the process of removing the waste from these tanks in order to place it into vitrified, stable state for longer term storage. The last stage in the removal sequence is a chemical cleaning step that breaks up and dissolves metal oxide solids that cannot be easily pumped out of the tank. Oxalic acid (OA) will be used to chemically clean the tanks after waste retrieval is completed. The waste tanks at SRS were constructed from carbon steel materials and thus are vulnerable to corrosion in acidic media. In addition to structural impacts, the impact of corrosion on the hydrogen generated during the process must be assessed. Electrochemical and coupon immersion tests were used to investigate the corrosion mechanism at anticipated process conditions. The testing showed that the corrosion rates were dependent upon the reduction of the iron species that had dissolved in solution. Initial corrosion rates were elevated due to the reduction of the ferric species to ferrous species. At later times, as the ferric species depleted, the corrosion rate decreased. On the other hand, the hydrogen evolution reaction became more dominant.

  7. Tsunami Amplification due to Focusing

    Moore, C. W.; Kanoglu, U.; Titov, V. V.; Aydin, B.; Spillane, M. C.; Synolakis, C. E.

    2012-12-01

    Tsunami runup measurements over the periphery of the Pacific Ocean after the devastating Great Japan tsunami of 11 March 2011 showed considerable variation in far-field and near-field impact. This variation of tsunami impact have been attributed to either directivity of the source or by local topographic effects. Directivity arguments alone, however, cannot explain the complexity of the radiated patterns in oceans with trenches and seamounts. Berry (2007, Proc. R. Soc. Lond. A 463, 3055-3071) discovered how such underwater features may concentrate tsunamis into cusped caustics and thus cause large local amplifications at specific focal points. Here, we examine focusing and local amplification, not by considering the effects of underwater diffractive lenses, but by considering the details of the dipole nature of the initial profile, and propose that certain regions of coastline are more at-risk, not simply because of directivity but because typical tsunami deformations create focal regions where abnormal tsunami wave height can be registered (Marchuk and Titov, 1989, Proc. IUGG/IOC International Tsunami Symposium, Novosibirsk, USSR). In this work, we present a new general analytical solution of the linear shallow-water wave equation for the propagation of a finite-crest-length source over a constant depth without any restriction on the initial profile. Unlike the analytical solution of Carrier and Yeh (2005, Comp. Mod. Eng. & Sci. 10(2), 113-121) which was restricted to initial conditions with Gaussian profiles and involved approximation, our solution is not only exact, but also general and allows the use of realistic initial waveform such as N-waves as defined by Tadepalli and Synolakis (1994, Proc. R. Soc. Lond. A 445, 99-112). We then verify our analytical solution for several typical wave profiles, both with the NOAA tsunami forecast model MOST (Titov and Synolakis, 1998, J. Waterw. Port Coast. Ocean Eng. 124(4), 157-171) which is validated and verified through

  8. By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

    Tolle, Fabian; Wilke, Julian; Wengel, Jesper;

    2014-01-01

    The selection of nucleic acid aptamers is an increasingly important approach to generate specific ligands binding to virtually any molecule of choice. However, selection-inherent amplification procedures are prone to artificial by-product formation that prohibits the enrichment of target-recogniz......The selection of nucleic acid aptamers is an increasingly important approach to generate specific ligands binding to virtually any molecule of choice. However, selection-inherent amplification procedures are prone to artificial by-product formation that prohibits the enrichment of target......-recognizing aptamers. Little is known about the formation of such by-products when employing nucleic acid libraries as templates. We report on the formation of two different forms of by-products, named ladder- and non-ladder-type observed during repetitive amplification in the course of in vitro selection experiments...... improving the success rate of aptamer selection....

  9. Performance Degradation Tests of Phosphoric Acid Doped Polybenzimidazole Membrane Based High Temperature Polymer Electrolyte Membrane Fuel Cells

    Zhou, Fan; Araya, Samuel Simon; Grigoras, Ionela;

    2015-01-01

    Degradation tests of two phosphoric acid (PA) doped PBI membrane based HT-PEM fuel cells were reported in this paper to investigate the effects of start/stop and the presence of methanol in the fuel to the performance degradation of the HT-PEM fuel cell. Continuous tests with pure dry H2 and meth......Degradation tests of two phosphoric acid (PA) doped PBI membrane based HT-PEM fuel cells were reported in this paper to investigate the effects of start/stop and the presence of methanol in the fuel to the performance degradation of the HT-PEM fuel cell. Continuous tests with pure dry H2...

  10. Field Operations Program Chevrolet S-10 (Lead-Acid) Accelerated Reliability Testing - Final Report

    J. Francfort (INEEL); J. Argueta; M. Wehrey (Southern California Edison); D. Karner; L. Tyree (Electric Transportation Applications)

    1999-07-01

    This report summarizes the Accelerated Reliability testing of five lead-acid battery-equipped Chevrolet S-10 electric vehicles by the US Department of Energy's Field Operations Program and the Program's testing partners, Electric Transportation Applications (ETA) and Southern California Edison (SCE). ETA and SCE operated the S-10s with the goal of placing 25,000 miles on each vehicle within 1 year, providing an accelerated life-cycle analysis. The testing was performed according to established and published test procedures. The S-10s' average ranges were highest during summer months; changes in ambient temperature from night to day and from season-to-season impacted range by as much as 10 miles. Drivers also noted that excessive use of power during acceleration also had a dramatic effect on vehicle range. The spirited performance of the S-10s created a great temptation to inexperienced electric vehicle drivers to ''have a good time'' and to fully utilize the S-10's acceleration capability. The price of injudicious use of power is greatly reduced range and a long-term reduction in battery life. The range using full-power accelerations followed by rapid deceleration in city driving has been 20 miles or less.

  11. Mechanisms of metal-induced centrosome amplification.

    Holmes, Amie L; Wise, John Pierce

    2010-12-01

    Exposure to toxic and carcinogenic metals is widespread; however, their mechanisms of action remain largely unknown. One potential mechanism for metal-induced carcinogenicity and toxicity is centrosome amplification. Here we review the mechanisms for metal-induced centrosome amplification, including arsenic, chromium, mercury and nano-titanium dioxide. PMID:21118148

  12. Mechanisms of Metal-Induced Centrosome Amplification

    Holmes, Amie L.; Wise, John Pierce

    2010-01-01

    Exposure to toxic and carcinogenic metals is widespread; however, their mechanisms of action remain largely unknown. One potential mechanism for metal-induced carcinogenicity and toxicity is centrosome amplification. Here, we review the mechanisms for metal-induced centrosome amplification, including arsenic, chromium, mercury and nano-titanium dioxide.

  13. Risk Perception and Social Amplification

    Smith, R.E. [Environment Agency (United Kingdom)

    2001-07-01

    This paper seeks to consider social amplification as it applies to risk perception. Perceptions of the magnitude of a risk are conditioned by issues such as the degree of uncertainty in probability and consequences, the nature of the consequences and the relative weightings placed on probability and consequences. Risk perceptions are also influenced by factors such as confidence in the operator of an industrial process, trust in the regulator and the perceived fairness of regulatory decision-making. Different people may hold different views about these issues and there may also be difficulties in communication. The paper identifies and discusses self-reinforcing mechanisms, which will be labelled 'lock-in' here. They appear to apply in many situations where social amplification is observed. Historically, the term 'lock-in' has been applied mainly in the technological context but, in this paper, four types of lock-in are identified, namely scientific/technological, economic, social and institutional lock-in. One type of lock-in tends to lead to the next and all are buttressed by people's general acceptance of the familiar, fear of the unknown and resistance to change. The regulator seeks to make decisions which achieve the common good rather than supporting or perpetuating any set of vested interests. In this regard the locked-in positions of stakeholders, whether organisations, interest groups, or individual members of the public, are obstacles and challenges. Existing methods of consultation are unsatisfactory in terms of achieving a proper and productive level of dialogue with stakeholders.

  14. Visual inspection with acetic acid as a cervical cancer test: accuracy validated using latent class analysis

    McGrath John A

    2007-07-01

    Full Text Available Abstract Background The purpose of this study was to validate the accuracy of an alternative cervical cancer test – visual inspection with acetic acid (VIA – by addressing possible imperfections in the gold standard through latent class analysis (LCA. The data were originally collected at peri-urban health clinics in Zimbabwe. Methods Conventional accuracy (sensitivity/specificity estimates for VIA and two other screening tests using colposcopy/biopsy as the reference standard were compared to LCA estimates based on results from all four tests. For conventional analysis, negative colposcopy was accepted as a negative outcome when biopsy was not available as the reference standard. With LCA, local dependencies between tests were handled through adding direct effect parameters or additional latent classes to the model. Results Two models yielded good fit to the data, a 2-class model with two adjustments and a 3-class model with one adjustment. The definition of latent disease associated with the latter was more stringent, backed by three of the four tests. Under that model, sensitivity for VIA (abnormal+ was 0.74 compared to 0.78 with conventional analyses. Specificity was 0.639 versus 0.568, respectively. By contrast, the LCA-derived sensitivity for colposcopy/biopsy was 0.63. Conclusion VIA sensitivity and specificity with the 3-class LCA model were within the range of published data and relatively consistent with conventional analyses, thus validating the original assessment of test accuracy. LCA probably yielded more likely estimates of the true accuracy than did conventional analysis with in-country colposcopy/biopsy as the reference standard. Colpscopy with biopsy can be problematic as a study reference standard and LCA offers the possibility of obtaining estimates adjusted for referent imperfections.

  15. Intergranular corrosion testing of austenitic stainless steels in nitric acid solutions

    Whillock, G.O.H.; Dunnett, B. F. [British Nuclear Fuels plc, BNFL, B170, Sellafield, Seascale, Cumbria CA20 1PG (United Kingdom)

    2004-07-01

    In hot strong nitric acid solutions, stainless steels exhibit intergranular corrosion. Corrosion rates are often measured from immersion testing of specimens manufactured from the relevant material (e.g. plate or pipe). The corrosion rates, measured from weight loss, are found to increase with time prior to reaching steady state, which can take thousands of hours to achieve. The apparent increase in corrosion rate as a function of time was found to be an artefact due to the surface area of the specimen's being used in the corrosion rate calculations, rather than that of the true area undergoing active corrosion i.e. the grain boundaries. The steady state corrosion rate coincided with the onset of stable grain dropping, where the use of the surface area of the specimen to convert the weight loss measurements to corrosion rates was found to be appropriate. This was confirmed by sectioning of the specimens and measuring the penetration depths. The rate of penetration was found to be independent of time and no induction period was observed. A method was developed to shorten considerably the testing time to reach the steady state corrosion rate by use of a pre-treatment that induces grain dropping. The long-term corrosion rates from specimens which were pre-treated was similar to that achieved after prolonged testing of untreated (i.e. initially ground) specimens. The presence of cut surfaces is generally unavoidable in the simple immersion testing of specimens in test solutions. However, inaccuracy in the results may occur as the measured corrosion rate is often influenced by the orientation of the microstructure, the highest rates typically being observed on the cut surfaces. Two methods are presented which allow deconvolution of the corrosion rates from immersion testing of specimens containing cut surfaces, thus allowing reliable prediction of the long-term corrosion rate of plate surfaces. (authors)

  16. Intergranular corrosion testing of austenitic stainless steels in nitric acid solutions

    In hot strong nitric acid solutions, stainless steels exhibit intergranular corrosion. Corrosion rates are often measured from immersion testing of specimens manufactured from the relevant material (e.g. plate or pipe). The corrosion rates, measured from weight loss, are found to increase with time prior to reaching steady state, which can take thousands of hours to achieve. The apparent increase in corrosion rate as a function of time was found to be an artefact due to the surface area of the specimen's being used in the corrosion rate calculations, rather than that of the true area undergoing active corrosion i.e. the grain boundaries. The steady state corrosion rate coincided with the onset of stable grain dropping, where the use of the surface area of the specimen to convert the weight loss measurements to corrosion rates was found to be appropriate. This was confirmed by sectioning of the specimens and measuring the penetration depths. The rate of penetration was found to be independent of time and no induction period was observed. A method was developed to shorten considerably the testing time to reach the steady state corrosion rate by use of a pre-treatment that induces grain dropping. The long-term corrosion rates from specimens which were pre-treated was similar to that achieved after prolonged testing of untreated (i.e. initially ground) specimens. The presence of cut surfaces is generally unavoidable in the simple immersion testing of specimens in test solutions. However, inaccuracy in the results may occur as the measured corrosion rate is often influenced by the orientation of the microstructure, the highest rates typically being observed on the cut surfaces. Two methods are presented which allow deconvolution of the corrosion rates from immersion testing of specimens containing cut surfaces, thus allowing reliable prediction of the long-term corrosion rate of plate surfaces. (authors)

  17. Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests.

    Argyri, Anthoula A; Zoumpopoulou, Georgia; Karatzas, Kimon-Andreas G; Tsakalidou, Effie; Nychas, George-John E; Panagou, Efstathios Z; Tassou, Chrysoula C

    2013-04-01

    The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes

  18. Retrieval and Amplification of DNA from Unstained Histopathological Sections

    DonnaC.MONTAGUE; BeverlyD.LYN-COOK; 等

    1993-01-01

    Testing of compounds for carcinogenic potential in vivo involves various experimental designs.A few of these techniques are directed to demonstrate the genotoxicity and mutagenicity of the compound by histopathology.These changes shown by histochemical means include monoclonal antibody directed cellular markers.Development of the polymerase chain reaction technique(PCR)for amplification of DNA has facilitated the investigation of molecular events related to the formation of malignant neoplasms.We describe here a method for screening tissues for mutations of the H-ras gene using monoclonal antibodies directed toward normal and mutant p21 proteins.Formalin-fixed,paraffinembedded tissue sections are used to subsequently confirm the gene mutation by PCR amplification of the H-ras gene.The results indicated a successful application of this technique to demonstrate the presence of p21 oncoprotein in the tissues tested.

  19. Quantification of HER2 autoantibodies in the amplification phenomenon of HER2 in breast cancer

    Lauterlein, Jens-Jacob L; Petersen, Eva R B; Olsen, Dorte Aa; Østergaard, Birthe; Brandslund, Ivan

    2011-01-01

    Gene amplification of HER2 (human epidermal growth factor receptor 2) is a well-known phenomenon in various cancers. However, little is known about the mechanism of the gene amplification phenomenon itself. Autoantibodies to cellular receptors have been described in several cancer types. We...... hypothesised that autoantibodies against HER2 might have a stimulatory capacity and could be the cause of the HER2 gene amplification phenomenon. To investigate this, we developed a test for the detection of autoantibodies against HER2 in serum (S-HER2Ab)....

  20. Risk Types and Risk Amplification of Online Finance

    Gujun Yan

    2013-01-01

    Online finance not only has the same risks as the traditional financial sector, but also brings new types of risks and the amplification effect of the financial risks. In the circumstances of online finance, risk correlation between countries is increasing due to worldwide mutual penetration of financial businesses and customers. Facing the risk test of the Internet finance, risk control and management must be strengthened in order to achieve its sustainable developments.

  1. Bioleaching of heavy metals from soil using fungal-organic acids : bench scale testing

    Cathum, S.J.; Ousmanova, D.; Somers, A.; Punt, M. [SAIC Canada, Ottawa, ON (Canada); Brown, C.E. [Environment Canada, Ottawa, ON (Canada). Emergencies Engineering Division]|[Environment Canada, Ottawa, ON (Canada). Environmental Technology Centre

    2006-07-01

    The ability of fungi to solubilize metals from solid materials may present new opportunities in environmental remediation. This paper presented details of a bench scale experiment that evaluated the leaching of heavy metals from contaminated soil using in situ fungal-generated organic acids. Rice was used as the growing media for organic acid production by A. foetidus. The cultivated fungus was placed on large pieces of potato-dextrose agar (PDA) plates and suspended in 5 L of sterilized water. The cooked rice was inoculated by pouring the 5 L spore suspension over the rice layer. Soil was obtained from a soil pile impacted with heavy metals at a private industrial site and augmented with Pb-contaminated soil. A polyethylene tub was used with a drain pipe leading to a leachate vessel. Crushed stone was spread over the bottom of the tub to assist leachate drainage. Approximately 45 kg of the contaminated soil was spread evenly over the stone layer to a depth of 10 cm. The concentrated spore suspension was sprinkled over the rice. Each week the leachate collection vessel was removed from the bioleaching system and the fine soil particles were allowed to settle. A control was run using the contaminated soil and solid substrate without fungus. Growth of A. foetidus was observed in both control experiment and test experiment after a period of 35 days. The pH of the leachate was measured as the fungal growth progressed. The process was assessed using ICP Mass Spectroscopy and electron spectroscopy, which showed that approximately 65 g of heavy metals were mobilized from 45 kg of soil, and that the biological leaching process resulted in greater mobilization of heavy metals relative to the control experiment. It was concluded that organic acids generated by A. foetidus were capable of leaching heavy metals from the soil. 30 refs., 4 tabs., 15 figs.

  2. Sildenafil enhances the peripheral antinociceptive effect of ellagic acid in the rat formalin test

    Mohammad Taghi Mansouri

    2014-01-01

    Full Text Available Objective: Ellagic acid (EA, a major polyphenolic compound of pomegranate juice, produces antinociceptive effects, which are mediated through opioidergic and nitric oxide-cyclic guanosine monophosphate (NO-cGMP pathways. The present study was conducted to elucidate the peripheral antinociceptive effect of EA alone and in combination with sildenafil in the rat formalin test. Materials and Methods: Pain was produced by intraplantar injection of formalin (2.5% in rats and nociceptive behavior was measured as the number of flinches every 5 min in 60 min after injection. Results: Local administration of EA and sildenafil dose-dependently increased the nociception threshold in both phases of the test. Moreover, sub-effective doses of sildenafil (25 or 50 mcg/paw, i.p. significantly and dose-dependently enhanced the antinociception induced by a sub-effective dose of EA (60 mcg/paw, i.pl. in both phases of the test. The antinociception produced by these drugs alone, or in combination, was due to a peripheral site of action, since the administration in the contralateral paw was ineffective. Conclusion: Our results suggest that EA has local peripheral antinociceptive activity, and enhancement of this effect with sildenafil probably occurs through the inhibition of cGMP metabolism.

  3. A quantitative test of infrared optical constants for supercooled sulphuric and nitric acid droplet aerosols

    R. Wagner

    2003-05-01

    Full Text Available In situ Fourier transform infrared (FTIR extinction spectra of supercooled H2SO4/H2O and HNO3/H2O solution droplets were recorded in the large coolable aerosol chamber AIDA (Aerosol Interactions and Dynamics in the Atmosphere of Forschungszentrum Karlsruhe for a range of aerosol compositions and at temperatures extending down to 192 K. The measured spectra were quantitatively analysed in terms of aerosol composition and mass concentration by using Mie theory in combination with published refractive index data as input parameters. Simultaneously, total sulphuric acid and nitric acid mass concentrations from filter analysis and total water concentrations measured with the Lyman-a hygrometer of Forschungszentrum Jülich were used to calculate the aerosol composition at thermodynamic equilibrium inside the aerosol chamber. By comparing these measured aerosol parameters with those retrieved from the analysis of the FTIR spectra, the accuracy of the literature data sets of refractive indices could be assessed. In summary, four data sets were tested in the H2SO4/H2O system as well as two data sets in the HNO3/H2O system, partly revealing significant discrepancies in the retrieved aerosol properties. Potential explanations for these differences are discussed in this paper.

  4. 75 FR 22814 - Guidance for Industry: Nucleic Acid Testing (NAT) for Human Immunodeficiency Virus Type 1 (HIV-1...

    2010-04-30

    ... (70 FR 43439), FDA announced the availability of the draft guidance of the same title. FDA received...: Nucleic Acid Testing (NAT) for Human Immunodeficiency Virus Type 1 (HIV-1) and Hepatitis C Virus (HCV... Immunodeficiency Virus Type 1 (HIV-1) and Hepatitis C Virus (HCV): Testing, Product Disposition, and Donor...

  5. Cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner by using MTT assay on culture cell line

    Christian Khoswanto

    2008-09-01

    Full Text Available Background: Open dentin is always covered by smear layer, therefore before restoration is performed, cavity or tooth which has been prepared should be clean from dirt. The researchers suggested that clean dentin surface would reach effective adhesion between resin and tooth structure, therefore dentin conditioner like citric acid was used to reach the condition. Even though citric acid is not strong acid but it can be very erosive to oral mucous. Several requirements should be fulfilled for dental product such as non toxic, non irritant, biocompatible and should not have negative effect against local, systemic or biological environment. Cytotoxicity test was apart of biomaterial evaluation and needed for standard screening. Purpose: This study was to know the cytotoxicity of 40, 50, 60% citric acid as dentin conditioner using MTT assay. Method: This study is an experimental research using the Post-Test Only Control Group Design. Six samples of each 40, 50 and 60% citric acid for citotoxicity test using MTT assay. The density of optic formazan indicated the number of living cells. All data were statistically analyzed by one way ANOVA. Result: The percentage of living cells in 40, 50 and 60% citric acid were 95.14%, 93.42% and 93.14%. Conclusion: Citric acid is non toxic and safe to be used as dentine conditioner.

  6. Marked heterogeneity of HER2/NEU gene amplification in endometrial serous carcinoma.

    Buza, Natalia; Hui, Pei

    2013-12-01

    Significant heterogeneity of HER2 protein expression has been recently observed in HER2 positive endometrial serous carcinomas. Tumor cells with HER2 overexpression and/or gene amplification in a heterogeneous tumor may represent a biologically more aggressive subclone that is clinically relevant to prognosis and potential targeted therapy. To correlate with HER2 protein heterogeneity, we investigated the heterogeneity of HER2/NEU gene amplification in endometrial serous carcinoma. A total of 17 endometrial serous carcinomas with heterogeneous HER2 protein expression were selected for the study, including nine cases with a 3+ and eight cases with a 2+ immunohistochemical score. Initial reflex HER2 FISH was available for seven of the eight 2+ cases, five of which showed HER2/NEU gene amplification. All 17 cases underwent repeat FISH targeting larger tumor tissue areas. Ten cases (72%) displayed striking heterogeneity of HER2/NEU gene copy number in the form of cluster amplification. Diffuse HER2 amplification was observed in four cases, no amplification was seen in three tumors. In cases with cluster amplification, HER2 protein overexpression by immunohistochemistry closely correlated at the cellular level with HER2/NEU gene amplification. In conclusion, the significant percentage of cases with heterogeneous HER2/NEU gene amplification indicates that the existing HER2 testing guidelines designed for breast cancer may not be applicable to endometrial serous carcinoma. Clinical testing on multiple different tumor samples or large tumor tissue sections is recommended for both immunohistochemistry and FISH assessment of HER2 status. Direct comparison with the HER2 immunostaining pattern may be helpful in detecting HER2 amplified areas in a heterogeneous tumor. PMID:24123408

  7. Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences

    La Mura Maurizio; Lee David; Allnutt Theo R; Powell Wayne

    2009-01-01

    Abstract Background The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. Results We have tested the specificity and sensitivity of the technique for use in GMO studies. Res...

  8. Detection of viable Giardia cysts by amplification of heat shock-induced mRNA.

    Abbaszadegan, M; Huber, M. S.; Gerba, C P; Pepper, I L

    1997-01-01

    Primers obtained from gene sequences coding for heat shock proteins (HSP) were used to specifically detect enteric protozoans of the genus Giardia. The HSP primers amplified Giardia DNA or the corresponding RNA sequences obtained from lysed cysts and gave a 163-bp product. Since the presence of the product did not indicate whether the cysts were viable, these amplifications are a presence/absence test only. In contrast, amplification of heat shock-induced mRNA utilizing the same HSP primers w...

  9. Influence of citric acid solution as a test drink in the 14C-urea breath test for diagnosis of helicobactor pylori infection

    Different test meals are used in the 14C-urea breath test (UBT) for the detection of H.pylori infection. The purpose of using test meals is to slow gastric emptying and to maximise the distribution of the urea substrate within the stomach so as to increase the area and time of contact between bacteria and substrate. Recently, citric acid has been suggested as an improved liquid test meal. The mechanism is not known and could act by delaying gastric emptying, decreasing the pH at the site of the bacteria, or both

  10. Quantum Amplitude Amplification and Estimation

    Brassard, G; Mosca, M; Tapp, A; Brassard, Gilles; Hoyer, Peter; Mosca, Michele; Tapp, Alain

    2000-01-01

    Consider a Boolean function $\\chi: X \\to \\{0,1\\}$ that partitions set $X$ between its good and bad elements, where $x$ is good if $\\chi(x)=1$ and bad otherwise. Consider also a quantum algorithm $\\mathcal A$ such that $A \\ket{0} = \\sum_{x\\in X} \\alpha_x \\ket{x}$ is a quantum superposition of the elements of $X$, and let $a$ denote the probability that a good element is produced if $A \\ket{0}$ is measured. If we repeat the process of running $A$, measuring the output, and using $\\chi$ to check the validity of the result, we shall expect to repeat $1/a$ times on the average before a solution is found. *Amplitude amplification* is a process that allows to find a good $x$ after an expected number of applications of $A$ and its inverse which is proportional to $1/\\sqrt{a}$, assuming algorithm $A$ makes no measurements. This is a generalization of Grover's searching algorithm in which $A$ was restricted to producing an equal superposition of all members of $X$ and we had a promise that a single $x$ existed such tha...