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Sample records for acid amplification testing

  1. Nucleic Acid Amplification Testing for Neisseria gonorrhoeae

    PubMed Central

    Whiley, David M.; Tapsall, John W.; Sloots, Theo P.

    2006-01-01

    Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review. PMID:16436629

  2. Nucleic Acid Amplification Testing in Suspected Child Sexual Abuse

    ERIC Educational Resources Information Center

    Esernio-Jenssen, Debra; Barnes, Marilyn

    2011-01-01

    The American Academy of Pediatrics recommends that site-specific cultures be obtained, when indicated, for sexually victimized children. Nucleic acid amplification testing is a highly sensitive and specific methodology for identifying sexually transmitted infections. Nucleic acid amplification tests are also less invasive than culture, and this…

  3. Towards Improved Accuracy of Bordetella pertussis Nucleic Acid Amplification Tests

    PubMed Central

    2012-01-01

    In many clinical microbiology laboratories, nucleic acid amplification tests such as PCR have become the routine methods for the diagnosis of pertussis. While PCR has greatly increased the ability of laboratories to detect Bordetella pertussis infections, it has also been associated with false-positive results that can, given the tendency of B. pertussis to cause outbreaks, result in unnecessary and costly control measures. The species specificity of Bordetella gene targets and their number of copies per genome greatly impact the performance characteristics of nucleic acid amplification tests for B. pertussis. It is crucial that laboratorians recognize these characteristics, to limit false-positive test results and prevent pseudo-outbreaks. PMID:22442315

  4. Multiplex Nucleic Acid Amplification Test for Diagnosis of Dengue Fever, Malaria, and Leptospirosis

    PubMed Central

    Waggoner, Jesse J.; Abeynayake, Janaki; Balassiano, Ilana; Lefterova, Martina; Sahoo, Malaya K.; Liu, Yuanyuan; Vital-Brazil, Juliana Magalhães; Gresh, Lionel; Balmaseda, Angel; Harris, Eva; Banaei, Niaz

    2014-01-01

    Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens. PMID:24671788

  5. Multiplex nucleic acid amplification test for diagnosis of dengue fever, malaria, and leptospirosis.

    PubMed

    Waggoner, Jesse J; Abeynayake, Janaki; Balassiano, Ilana; Lefterova, Martina; Sahoo, Malaya K; Liu, Yuanyuan; Vital-Brazil, Juliana Magalhães; Gresh, Lionel; Balmaseda, Angel; Harris, Eva; Banaei, Niaz; Pinsky, Benjamin A

    2014-06-01

    Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.

  6. Clinical impact of switching conventional enzyme immunoassay with nucleic acid amplification test for suspected Clostridium difficile-associated diarrhea.

    PubMed

    Johnson, Steven W; Kanatani, Meganne; Humphries, Romney M; Uslan, Daniel Z

    2013-04-01

    The impact of a new Clostridium difficile nucleic acid amplification test (NAAT) on antibiotic utilization in patients with suspected C difficile infection was assessed. This single-center, cross-sectional study of 270 patients demonstrated that the use of NAAT decreased antibiotic expenditure by reducing prolonged empiric days of therapy in these patients.

  7. Use of Nucleic Acid Amplification Tests in Tuberculosis Patients in California, 2010–2013

    PubMed Central

    Peralta, Gianna; Barry, Pennan

    2016-01-01

    Background. Nucleic acid amplification tests (NAATs) have been used as a diagnostic tool for tuberculosis (TB) in the United States for many years. We sought to assess NAAT use in TB patients in California during a period of time when NAAT availability increased throughout the world. Methods. We conducted a retrospective review of surveillance data from 6051 patients with culture-confirmed pulmonary TB who were reported to the California TB registry during 2010–2013. Results. Only 2336 of 6051 (39%) TB patients had a NAAT for diagnosis before culture results. Although 90% (N = 2101) with NAAT had positive test results, 9% (N = 217) had falsely negative NAAT results, and 0.8% (N = 18) had indeterminate NAAT results. The median time from specimen collection to TB treatment initiation was shorter when NAAT was used (3 vs 14 days, P < .0001), and patients with a positive NAAT result initiated treatment earlier than patients with a falsely negative result (1 vs 11 days from NAAT report, P < .0001). We confirmed the increased sensitivity of NAAT compared with acid-fast bacilli (AFB) smear microscopy in our study population; 92 of 145 AFB smear-negative patients had positive NAATs. Median time from specimen collection to NAAT result report differed by health jurisdiction, from 1 to 11 working days. Conclusions. Increased use of NAATs in diagnosis of pulmonary TB could decrease the time-to-treatment initiation and consequently decrease transmission. However, differential use and access to NAAT may prevent full realization of NAAT benefits in California. PMID:27957506

  8. Isothermal Amplification of Nucleic Acids.

    PubMed

    Zhao, Yongxi; Chen, Feng; Li, Qian; Wang, Lihua; Fan, Chunhai

    2015-11-25

    Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

  9. Lyophilized Visually Readable Loop-Mediated Isothermal Reverse Transcriptase Nucleic Acid Amplification Test for Detection Ebola Zaire RNA.

    PubMed

    Carter, Christoph; Akrami, Kevan; Hall, Drew; Smith, Davey; Aronoff-Spencer, Eliah

    2017-02-24

    Recent viral outbreaks highlight the need for reliable, yet broadly deployable diagnostics for detection of epidemic and emerging pathogens. In this study we designed and optimized methods to visually detect viral nucleic acid by isothermal amplification and SYBR dye intercalation. We designed and tested loop-mediated isothermal amplification (LAMP) primers and lyophilized reactions to optimize the detection of Zaire Ebola Virus (ZEBOV) and further evolved the LAMP platform to allow room-temperature storage for deployment in resource limited settings. Our results demonstrated excellent sensitivity and specificity for viral nucleic acid sequences with lower limits of detection of less than 100 copies. Moreover, lyophilized reaction mixtures retained activity for prolonged periods under dry conditions at room temperature. This approach offers a way for detection of emerging viruses in resource limited settings.

  10. Relative Accuracy of Nucleic Acid Amplification Tests and Culture in Detecting Chlamydia in Asymptomatic Men

    PubMed Central

    Cheng, Hong; Macaluso, Maurizio; Vermund, Sten H.; Hook, Edward W.

    2001-01-01

    Published estimates of the sensitivity and specificity of PCR and ligase chain reaction (LCR) for detecting Chlamydia trachomatis are potentially biased because of study design limitations (confirmation of test results was limited to subjects who were PCR or LCR positive but culture negative). Relative measures of test accuracy are less prone to bias in incomplete study designs. We estimated the relative sensitivity (RSN) and relative false-positive rate (RFP) for PCR and LCR versus cell culture among 1,138 asymptomatic men and evaluated the potential bias of RSN and RFP estimates. PCR and LCR testing in urine were compared to culture of urethral specimens. Discordant results (PCR or LCR positive, but culture negative) were confirmed by using a sequence including the other DNA amplification test, direct fluorescent antibody testing, and a DNA amplification test to detect chlamydial major outer membrane protein. The RSN estimates for PCR and LCR were 1.45 (95% confidence interval [CI] = 1.3 to 1.7) and 1.49 (95% CI = 1.3 to 1.7), respectively, indicating that both methods are more sensitive than culture. Very few false-positive results were found, indicating that the specificity levels of PCR, LCR, and culture are high. The potential bias in RSN and RFP estimates were <5 and <20%, respectively. The estimation of bias is based on the most likely and probably conservative parameter settings. If the sensitivity of culture is between 60 and 65%, then the true sensitivity of PCR and LCR is between 90 and 97%. Our findings indicate that PCR and LCR are significantly more sensitive than culture, while the three tests have similar specificities. PMID:11682509

  11. Gonorrhoea Diagnostic and Treatment Uncertainties: Risk Factors for Culture Negative Confirmation after Positive Nucleic Acid Amplification Tests.

    PubMed

    Vyth, Rebecka; Leval, Amy; Eriksson, Björn; Ericson, Eva-Lena; Marions, Lena; Hergens, Maria-Pia

    2016-01-01

    Gonorrhoea incidence has increased substantially in Stockholm during the past years. These increases have coincided with changes in testing practice from solely culture-based to nucleic acid amplification tests (NAAT). Gonorrhoea NAAT is integrated with Chlamydia trachomatis testing and due to opportunistic screening for chlamydia, testing prevalence for gonorrhoea has increased substantially in the Stockholm population. The aim of this study was to examine epidemiological risk-factors for discordant case which are NAAT positive but culture negative. These discordant cases are especially problematic as they give rise to diagnostic and treatment uncertainties with risk for subsequent sequelae. All gonorrhoea cases from Stockholm county during 2011-2012 with at least one positive N. gonorrhoea NAAT test and follow-up cultures were included (N = 874). Data were analysed using multivariate and stratified logistic regression models. Results showed that women were 4-times more likely (OR 4.9; 95% CI 2.4-6.7) than men to have discordant cultures. Individuals tested for gonorrhoea without symptoms were 2.3 times more likely (95% CI 1.5-3.5) than those with symptoms to be discordant. NAAT method and having one week or more between NAAT and culture testing were also indicative of an increased likelihood for discordance. Using NAAT should be based on proper clinical or epidemiological indications and, when positive, followed-up with a culture-based test within one week if possible. Routine gonorrhoea testing is not recommended in low prevalence populations.

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

    PubMed

    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.

  13. Evaluation of Six Commercial Nucleic Acid Amplification Tests for Detection of Neisseria gonorrhoeae and Other Neisseria Species▿

    PubMed Central

    Tabrizi, Sepehr N.; Unemo, Magnus; Limnios, Athena E.; Hogan, Tiffany R.; Hjelmevoll, Stig-Ove; Garland, Susanne M.; Tapsall, John

    2011-01-01

    Molecular detection of Neisseria gonorrhoeae in extragenital samples may result in false-positive results due to cross-reaction with commensal Neisseria species or Neisseria meningitidis. This study examined 450 characterized clinical culture isolates, comprising 216 N. gonorrhoeae isolates and 234 isolates of nongonococcal Neisseria species (n = 218) and 16 isolates of other closely related bacteria, with six commercial nucleic acid amplification tests (NAATs). The six NAATs tested were Gen-Probe APTIMA COMBO 2 and APTIMA GC, Roche COBAS Amplicor CT/NG and COBAS 4800 CT/NG tests, BD ProbeTec GC Qx amplified DNA assay, and Abbott RealTime CT/NG test. All assays except COBAS Amplicor CT/NG test where four (1.9%) isolates were not detected showed a positive result with all N. gonorrhoeae isolates (n = 216). Among the 234 nongonococcal isolates examined, initial results from all assays displayed some false-positive results due to cross-reactions. Specifically, the COBAS Amplicor and ProbeTec tests showed the highest number of false-positive results, detecting 33 (14.1%) and 26 (11%) nongonococcal Neisseria isolates, respectively. On the first testing, APTIMA COMBO 2, APTIMA GC, Abbott RealTime, and Roche COBAS 4800 showed lower level of cross-reactions with five (2.1%), four (1.7%), two (1%), and two (1%) of the isolates showing low-level positivity, respectively. Upon retesting of these nine nongonococcal isolates using freshly cultured colonies, none were positive by the APTIMA COMBO 2, Abbott RealTime, or COBAS 4800 test. In conclusion, the COBAS Amplicor and ProbeTec tests displayed high number of false-positive results, while the remaining NAATs showed only sporadic low-level false-positive results. Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites. PMID:21813721

  14. Non-instrumented nucleic acid amplification assay

    NASA Astrophysics Data System (ADS)

    Weigl, Bernhard H.; Domingo, Gonzalo; Gerlach, Jay; Tang, Dennis; Harvey, Darrel; Talwar, Nick; Fichtenholz, Alex; van Lew, Bill; LaBarre, Paul

    2008-02-01

    We have developed components of a diagnostic disposable platform that has the dual purpose of providing molecular diagnostics at the point of care (POC) as well as stabilizing specimens for further analysis via a centralized surveillance system. This diagnostic is targeted for use in low-resource settings by minimally trained health workers. The disposable device does not require any additional instrumentation and will be almost as rapid and simple to use as a lateral flow strip test - yet will offer the sensitivity and specificity of nucleic acid amplification tests (NAATs). The low-cost integrated device is composed of three functional components: (1) a sample-processing subunit that generates clean and stabilized DNA from raw samples containing nucleic acids, (2) a NA amplification subunit, and (3) visual amplicon detection sub-unit. The device integrates chemical exothermic heating, temperature stabilization using phase-change materials, and isothermal nucleic acid amplification. The aim of developing this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where there is no access to instrumentation. If a disease occurs, patients would be tested with the disposable in the field. A nucleic acid sample would be preserved within the spent disposable which could be sent to a central laboratory facility for further analysis if needed.

  15. Genomic detection of human immunodeficiency virus (HIV) by nucleic acid amplification test in a frequent platelet donor during the pre-seroconversion period.

    PubMed

    Pondé, Robério Amorim de Almeida

    2011-11-01

    Since serological donor-screening tests for HIV were introduced in 1985, the safety of donated blood components has improved dramatically. However, these tests do not completely prevent the risk of transfusion-associated HIV infection related to the use of blood donated during the pre-seroconversion window period. Testing based on nucleic acid amplification is being implemented to screen for HIV-infected blood donated during this period, which has reduced the probability of transmitting HIV through transfusion by shortening the window period. This article describes a case of acute HIV-1 infection, detected using a nucleic acid amplification test (NAT) in a repeat blood donor who donated during the pre-seroconversion window period and whose antigen and anti-HIV antibody expression was observed after molecular marker detection. In addition, the possible route of infection is discussed based on the patient's history, and finally, the need for NAT technology for blood donor screening is emphasized.

  16. Nucleic Acid Amplification Testing and Sequencing Combined with Acid-Fast Staining in Needle Biopsy Lung Tissues for the Diagnosis of Smear-Negative Pulmonary Tuberculosis

    PubMed Central

    Tian, Panwen; Chen, Xuerong; Liang, Zongan

    2016-01-01

    Background Smear-negative pulmonary tuberculosis (PTB) is common and difficult to diagnose. In this study, we investigated the diagnostic value of nucleic acid amplification testing and sequencing combined with acid-fast bacteria (AFB) staining of needle biopsy lung tissues for patients with suspected smear-negative PTB. Methods Patients with suspected smear-negative PTB who underwent percutaneous transthoracic needle biopsy between May 1, 2012, and June 30, 2015, were enrolled in this retrospective study. Patients with AFB in sputum smears were excluded. All lung biopsy specimens were fixed in formalin, embedded in paraffin, and subjected to acid-fast staining and tuberculous polymerase chain reaction (TB-PCR). For patients with positive AFB and negative TB-PCR results in lung tissues, probe assays and 16S rRNA sequencing were used for identification of nontuberculous mycobacteria (NTM). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy of PCR and AFB staining were calculated separately and in combination. Results Among the 220 eligible patients, 133 were diagnosed with TB (men/women: 76/57; age range: 17–80 years, confirmed TB: 9, probable TB: 124). Forty-eight patients who were diagnosed with other specific diseases were assigned as negative controls, and 39 patients with indeterminate final diagnosis were excluded from statistical analysis. The sensitivity, specificity, PPV, NPV, and accuracy of histological AFB (HAFB) for the diagnosis of smear-negative were 61.7% (82/133), 100% (48/48), 100% (82/82), 48.5% (48/181), and 71.8% (130/181), respectively. The sensitivity, specificity, PPV, and NPV of histological PCR were 89.5% (119/133), 95.8% (46/48), 98.3% (119/121), and 76.7% (46/60), respectively, demonstrating that histological PCR had significantly higher accuracy (91.2% [165/181]) than histological acid-fast staining (71.8% [130/181]), P < 0.001. Parallel testing of histological AFB

  17. Nucleic acid amplification tests (polymerase chain reaction, ligase chain reaction) for the diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae in pediatric emergency medicine.

    PubMed

    Corneli, Howard M

    2005-04-01

    Nucleic acid amplification tests, such as ligase chain reaction and polymerase chain reaction, offer potential advantages of speed, simplicity, and accuracy in the detection of genitourinary tract infection with Neisseria gonorrhoeae and Chlamydia trachomatis. Their appropriate use in pediatric emergency medicine depends on an understanding of their strengths and weaknesses. Problems arise in defining the sensitivity and, especially, specificity of these tests. The clinical scenario, the site of infection, the age and sex of the patient, and especially the presence or absence of medicolegal concerns strongly affect the applicability of these tests. The risk of false positives may be significant even when legal concerns do not arise and even if a highly specific test is used. This article reviews the uses and limitations of such tests in pediatric emergency medicine. Discussion is directed to both technical and practical considerations.

  18. Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP)

    PubMed Central

    Poole, Catherine B.; Li, Zhiru; Alhassan, Andy; Guelig, Dylan; Diesburg, Steven; Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C.; LaBarre, Paul; Wanji, Samuel; Burton, Robert A.; Carlow, Clotilde K. S.

    2017-01-01

    Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs. PMID:28199317

  19. Amplification of trace amounts of nucleic acids

    DOEpatents

    Church, George M.; Zhang, Kun

    2008-06-17

    Methods of reducing background during amplification of small amounts of nucleic acids employ careful analysis of sources of low level contamination. Ultraviolet light can be used to reduce nucleic acid contaminants in reagents and equipment. "Primer-dimer" background can be reduced by judicious design of primers. We have shown clean signal-to-noise with as little as starting material as one single human cell (.about.6 picogram), E. coli cell (.about.5 femtogram) or Prochlorococcus cell (.about.3 femtogram).

  20. Detecting asymptomatic Trichomonas vaginalis in females using the BD ProbeTec™ Trichomonas vaginalis Q(x) nucleic acid amplification test.

    PubMed

    Lord, Emily; Newnham, Tana; Dorrell, Lucy; Jesuthasan, Gerald; Clarke, Lorraine; Jeffery, Katie; Sherrard, Jackie

    2017-03-01

    Trichomonas vaginalis (TV) rates in women are increasing and many are asymptomatic. Nucleic acid amplification tests (NAATs) are becoming the 'gold standard' for diagnosis. We aimed to establish our asymptomatic TV rates by testing all women attending Oxfordshire's Sexual Health service, regardless of symptoms, using the BD ProbeTec™ TV Q(x) NAATs (BDQ(x)). During BDQ(x)'s verification process, the sensitivity and specificity were calculated using results of 220 endocervical samples from symptomatic women, compared with culture. BDQ(x) was subsequently implemented and prospectively evaluated over 6 months in female attendees. Wet mount microscopy was also performed in symptomatics. Demographic and clinical characteristics of those diagnosed were analysed. From 220 samples tested by BDQ(x) and culture: 5 were positive on both and one solely using BDQ(x), giving a sensitivity and specificity of 100% and 99.53%, respectively. In the prospective cohort, of 5775 BDQ(x) tests, 33 (0.57%) were positive. 11/33 (33%) patients were asymptomatic. All patients diagnosed had risk factors: age >25 years (85%), residence in a deprived area (79%) and black ethnicity (21%). Despite BDQ(x) being highly sensitive and specific, with our low TV prevalence universal screening may not be justified. Targeted screening using local demographic data merits further investigation.

  1. Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

    PubMed

    Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

    2016-10-01

    Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis.

  2. Bioanalytical applications of isothermal nucleic acid amplification techniques.

    PubMed

    Deng, Huimin; Gao, Zhiqiang

    2015-01-01

    The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique.

  3. [Viral safety of biologicals: evaluation of hepatitis C virus (HCV) nucleic acid amplification test (NAT) assay and development of concentration method of HCV for sensitive detection by NAT].

    PubMed

    Uchida, Eriko; Yamaguchi, Teruhide

    2010-02-01

    The most important issue for the safety of biological products and blood products derived from human sources is how to prevent transmission of infectious agents. The hepatitis C virus (HCV) is a major public health problem due to its high prevalence. HCV is mainly transmitted by exposure to blood and highly infectious during the early window period with extremely low viral loads. Therefore it is important to develop more sensitive detection methods for HCV. In the case of blood products, both serological test and nucleic acid amplification test (NAT) are required to detect HCV. Since NAT is highly sensitive, establishment of a new standard is required for validation of NAT assay. NAT guideline and establishment of the standard for HCV RNA and HCV genotype panel is introduced in this review. On the other hand, to enhance the sensitivity of virus detection by NAT, a novel viral concentration method using polyethyleneimine (PEI)-conjugated magnetic beads (PEI beads) was developed. PEI beads concentration method is applicable to a wide range of viruses including HCV. Studies using the national standard for HCV RNA, HCV genotype panel and seroconversion panel, suggest that virus concentration method using PEI-beads is useful for improvement of the sensitivity of HCV detection by NAT and applicable to donor screening for HCV.

  4. Can mailed swab samples be dry-shipped for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis by nucleic acid amplification tests?

    PubMed

    Gaydos, Charlotte A; Farshy, Carol; Barnes, Mathilda; Quinn, Nicole; Agreda, Patricia; Rivers, Charles A; Schwebke, Jane; Papp, John

    2012-05-01

    Dry-shipped and mailed vaginal swabs collected at home have been used in research studies for the detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC), and Trichomonas vaginalis (TV) by nucleic acid amplification tests (NAATs) in screening programs. A verification study was performed to compare the limit of detection of CT, GC, and TV on swabs that were dry-shipped to paired swabs that were wet-shipped in transport media through the US mail. The Centers for Disease Control and Prevention prepared inocula in sterile water to mock simulated urogenital swabs with high to low concentrations of CT and GC. Replicate swabs were inoculated with 100 μL of dilutions and were dry transported or placed into commercial transport media ("wet") for mailing for NAAT testing. The University of Alabama prepared replicate concentrations of TV, which were similarly shipped and tested by NAAT. All paired dry and wet swabs were detectable for CT. For GC, all paired dry and wet swabs were detectable for GC at concentrations ≥ 10(3). At 10(2) and 10 CFU/mL, the 10 replicate GC results were variably positive. For TV, wet and dry shipped concentrations >10(2) TV/mL tested positive, while results at 10 TV/mL were negative for dry swabs. Holding replicate dry swabs at 55 (○)C 5 days before testing did not affect results. NAATs were able to detect CT, GC, and TV on dry transported swabs. Using NAATs for testing home-collected, urogenital swabs mailed in a dry state to a laboratory may be useful for outreach screening programs.

  5. Can mailed swab samples be dry-shipped for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis by nucleic acid amplification tests?

    PubMed Central

    Gaydos, Charlotte A.; Farshy, Carol; Barnes, Mathilda; Quinn, Nicole; Agreda, Patricia; Rivers, Charles A.; Schwebke, Jane; Papp, John

    2012-01-01

    Background Dry-shipped and mailed vaginal swabs collected at home have been used in research studies for the detection of C. trachomatis (CT), N. gonorrhoeae (GC), and Trichomonas vaginalis (TV) by nucleic acid amplification tests (NAATs) in screening programs. A verification study was performed to compare the limit of detection of CT, GC, and TV on swabs that were dry-shipped to paired swabs that were wet-shipped in transport media through the U.S. mail. Methods The Centers for Disease Control and Prevention prepared inocula in sterile water to mock simulated urogenital swabs with high to low concentrations of CT and GC. Replicate swabs were inoculated with 100µl of dilutions, were dry transported or placed into commercial transport media (“wet”) for mailing for NAAT testing. The University of Alabama prepared replicate concentrations of TV, which were similarly shipped and tested by NAAT. Results All paired dry and wet swabs were detectable for CT. For GC, all paired dry and wet swabs were detectable for GC at concentrations ≥103. At 102 and 10 CFU/ml, the 10 replicate GC results were variably positive. For TV, wet and dry shipped concentrations > 102 TV/ml tested positive, while results at 10 TV/ml were negative for dry swabs. Holding replicate dry swabs at 55°C 5 days before testing did not affect results. Conclusion NAATs were able to detect CT, GC, and TV on dry transported swabs. Using NAATs for testing home-collected, urogenital swabs mailed in a dry state to a laboratory may be useful for outreach screening programs. PMID:22578934

  6. Comparison of the Luminex xTAG respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections.

    PubMed

    Pabbaraju, Kanti; Tokaryk, Kara L; Wong, Sallene; Fox, Julie D

    2008-09-01

    Detection of respiratory viruses using sensitive real-time nucleic acid amplification tests (NATs) is invaluable for patient and outbreak management. However, the wide range of potential respiratory virus pathogens makes testing using individual real-time NATs expensive and laborious. The objective of this study was to compare the detection of respiratory virus targets using the Luminex xTAG respiratory viral panel (RVP) assay with individual real-time NATs used at the Provincial Laboratory of Public Health, Calgary, Alberta, Canada. The study included 1,530 specimens submitted for diagnosis of respiratory infections from December 2006 to May 2007. Direct-fluorescent-antigen-positive nasopharyngeal samples were excluded from this study. A total of 690 and 643 positives were detected by RVP and in-house NATs, respectively. Kappa correlation between in-house NATs and RVP for all targets ranged from 0.721 to 1.000. The majority of specimens missed by in-house NATs (96.7%) were positive for picornaviruses. Samples missed by RVP were mainly positive for adenovirus (51.7%) or respiratory syncytial virus (27.5%) by in-house NATs and in general had low viral loads. RVP allows for multiplex detection of 20 (and differentiation between 19) respiratory virus targets with considerable time and cost savings compared with alternative NATs. Although this first version of the RVP assay has lower sensitivity than in-house NATs for detection of adenovirus, it has good sensitivity for other targets. The identification of picornaviruses and coronaviruses and concurrent typing of influenza A virus by RVP, which are not currently included in our diagnostic testing algorithm, will improve our diagnosis of respiratory tract infections.

  7. Ligation with nucleic acid sequence-based amplification.

    PubMed

    Ong, Carmichael; Tai, Warren; Sarma, Aartik; Opal, Steven M; Artenstein, Andrew W; Tripathi, Anubhav

    2012-01-01

    This work presents a novel method for detecting nucleic acid targets using a ligation step along with an isothermal, exponential amplification step. We use an engineered ssDNA with two variable regions on the ends, allowing us to design the probe for optimal reaction kinetics and primer binding. This two-part probe is ligated by T4 DNA Ligase only when both parts bind adjacently to the target. The assay demonstrates that the expected 72-nt RNA product appears only when the synthetic target, T4 ligase, and both probe fragments are present during the ligation step. An extraneous 38-nt RNA product also appears due to linear amplification of unligated probe (P3), but its presence does not cause a false-positive result. In addition, 40 mmol/L KCl in the final amplification mix was found to be optimal. It was also found that increasing P5 in excess of P3 helped with ligation and reduced the extraneous 38-nt RNA product. The assay was also tested with a single nucleotide polymorphism target, changing one base at the ligation site. The assay was able to yield a negative signal despite only a single-base change. Finally, using P3 and P5 with longer binding sites results in increased overall sensitivity of the reaction, showing that increasing ligation efficiency can improve the assay overall. We believe that this method can be used effectively for a number of diagnostic assays.

  8. Nucleic acid amplification: Alternative methods of polymerase chain reaction

    PubMed Central

    Fakruddin, Md; Mannan, Khanjada Shahnewaj Bin; Chowdhury, Abhijit; Mazumdar, Reaz Mohammad; Hossain, Md. Nur; Islam, Sumaiya; Chowdhury, Md. Alimuddin

    2013-01-01

    Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically. PMID:24302831

  9. Nucleic acid amplification: Alternative methods of polymerase chain reaction.

    PubMed

    Fakruddin, Md; Mannan, Khanjada Shahnewaj Bin; Chowdhury, Abhijit; Mazumdar, Reaz Mohammad; Hossain, Md Nur; Islam, Sumaiya; Chowdhury, Md Alimuddin

    2013-10-01

    Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

  10. Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

    PubMed

    Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

    2016-04-01

    Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

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

    PubMed

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

    2015-10-20

    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.

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

    PubMed Central

    Mauk, Michael G.; Liu, Changchun; Song, Jinzhao; 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 (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. PMID:27600235

  13. Replica amplification of nucleic acid arrays

    DOEpatents

    Church, George M.

    2002-01-01

    A method of producing a plurality of a nucleic acid array, comprising, in order, the steps of amplifying in situ nucleic acid molecules of a first randomly-patterned, immobilized nucleic acid array comprising a heterogeneous pool of nucleic acid molecules affixed to a support, transferring at least a subset of the nucleic acid molecules produced by such amplifying to a second support, and affixing the subset so transferred to the second support to form a second randomly-patterned, immobilized nucleic acid array, wherein the nucleic acid molecules of the second array occupy positions that correspond to those of the nucleic acid molecules from which they were amplified on the first array, so that the first array serves as a template to produce a plurality, is disclosed.

  14. Increased amplification success from forensic samples with locked nucleic acids.

    PubMed

    Ballantyne, Kaye N; van Oorschot, Roland A H; Mitchell, R John

    2011-08-01

    Inadequate sample quantities and qualities can commonly result in poor DNA amplification success rates for forensic case samples. In some instances, modifying the PCR protocol or components may assist profiling by overcoming inhibition, or reducing the threshold required for successful amplification and detection. Incorporation of locked nucleic acids (LNAs) into PCR primers has previously been shown to increase amplification success for a range of non-forensic sample types and applications. To investigate their use in a forensic context, the PCR primers for four commonly used STR loci have been redesigned to include LNA bases. The modified LNA primers provided significantly increased amplification success when compared to standard DNA primers, with both high-quality buccal samples and simulated forensic casework samples. Peak heights increased by as much as 5.75× for the singleplex amplifications. When incorporated into multiplexes, the LNA primers continued to outperform standard DNA primers, with increased ease of optimisation, and increased amplification success. The use of LNAs in PCR primers can greatly assist the profiling of a range of samples, and increase success rates from challenging forensic samples.

  15. Replica amplification of nucleic acid arrays

    DOEpatents

    Church, George M.; Mitra, Robi D.

    2010-08-31

    Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.

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

    PubMed

    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.

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

    PubMed Central

    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

  18. Nucleic acid amplification using modular branched primers

    DOEpatents

    Ulanovsky, Levy; Raja, Mugasimangalam C.

    2001-01-01

    Methods and compositions expand the options for making primers for use in amplifying nucleic acid segments. The invention eliminates the step of custom synthesis of primers for Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially mimics the performance of a conventional, custom-made primer by matching the sequence of the priming site in the template. Each oligonucleotide module has a segment that matches one of the stretches within the priming site.

  19. A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection.

    PubMed

    Tang, Ruihua; Yang, Hui; Gong, Yan; You, MinLi; Liu, Zhi; Choi, Jane Ru; Wen, Ting; Qu, Zhiguo; Mei, Qibing; Xu, Feng

    2017-03-29

    Nucleic acid testing (NAT) has been widely used for disease diagnosis, food safety control and environmental monitoring. At present, NAT mainly involves nucleic acid extraction, amplification and detection steps that heavily rely on large equipment and skilled workers, making the test expensive, time-consuming, and thus less suitable for point-of-care (POC) applications. With advances in paper-based microfluidic technologies, various integrated paper-based devices have recently been developed for NAT, which however require off-chip reagent storage, complex operation steps and equipment-dependent nucleic acid amplification, restricting their use for POC testing. To overcome these challenges, we demonstrate a fully disposable and integrated paper-based sample-in-answer-out device for NAT by integrating nucleic acid extraction, helicase-dependent isothermal amplification and lateral flow assay detection into one paper device. This simple device allows on-chip dried reagent storage and equipment-free nucleic acid amplification with simple operation steps, which could be performed by untrained users in remote settings. The proposed device consists of a sponge-based reservoir and a paper-based valve for nucleic acid extraction, an integrated battery, a PTC ultrathin heater, temperature control switch and on-chip dried enzyme mix storage for isothermal amplification, and a lateral flow test strip for naked-eye detection. It can sensitively detect Salmonella typhimurium, as a model target, with a detection limit of as low as 10(2) CFU ml(-1) in wastewater and egg, and 10(3) CFU ml(-1) in milk and juice in about an hour. This fully disposable and integrated paper-based device has great potential for future POC applications in resource-limited settings.

  20. Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory.

    PubMed Central

    Ieven, M; Goossens, H

    1997-01-01

    Clinical laboratories are increasingly receiving requests to perform nucleic acid amplification tests for the detection of a wide variety of infectious agents. In this paper, the efficiency of nucleic acid amplification techniques for the diagnosis of respiratory tract infections is reviewed. In general, these techniques should be applied only for the detection of microorganisms for which available diagnostic techniques are markedly insensitive or nonexistent or when turnaround times for existing tests (e.g., viral culture) are much longer than those expected with amplification. This is the case for rhinoviruses, coronaviruses, and hantaviruses causing a pulmonary syndrome, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Coxiella burnetii. For Legionella spp. and fungi, contamination originating from the environment is a limiting factor in interpretation of results, as is the difficulty in differentiating colonization and infection. Detection of these agents in urine or blood by amplification techniques remains to be evaluated. In the clinical setting, there is no need for molecular diagnostic tests for the diagnosis of Pneumocystis carinii. At present, amplification methods for Mycobacterium tuberculosis cannot replace the classical diagnostic techniques, due to their lack of sensitivity and the absence of specific internal controls for the detection of inhibitors of the reaction. Also, the results of interlaboratory comparisons are unsatisfactory. Furthermore, isolates are needed for susceptibility studies. Additional work remains to be done on sample preparation methods, comparison between different amplification methods, and analysis of results. The techniques can be useful for the rapid identification of M. tuberculosis in particular circumstances, as well as the rapid detection of most rifampin-resistant isolates. The introduction of diagnostic amplification techniques into a clinical laboratory implies a level of proficiency for

  1. Head-to-head comparison of second-generation nucleic acid amplification tests for detection of Chlamydia trachomatis and Neisseria gonorrhoeae on urine samples from female subjects and self-collected vaginal swabs.

    PubMed

    Chernesky, Max; Jang, Dan; Gilchrist, Jodi; Hatchette, Todd; Poirier, André; Flandin, Jean-Frederic; Smieja, Marek; Ratnam, Sam

    2014-07-01

    In a comparison of 4 second-generation nucleic acid amplification tests performed with self-collected vaginal swab (SCVS) and first-void urine (FVU) specimens from 575 women, SCVS specimens indicated more infections than did FVU specimens in all assays. The prevalence rates were 9% (53/575 patients) for Chlamydia trachomatis and 2% (11/575 patients) for Neisseria gonorrhoeae. The clinical sensitivities for testing SCVS specimens for C. trachomatis were 98.1% on a Tigris system and 96.2% on a Panther system for the Aptima Combo 2 assay (Hologic Gen-Probe), 98.0% for the RealTime CT/NG assay on an m2000 instrument (Abbott), 90.6% for the ProbeTec CT/GC Q(x) assay on the Viper system (Becton Dickinson), and 84.6% for the cobas CT/NG assay on the cobas 4800 platform (Roche). Clinical sensitivities for C. trachomatis in FVU specimens were 88.7% (Tigris) and 88.0% (Panther) for the Aptima Combo 2 assay, 76.9% for the RealTime CT/NG assay, 75.5% for the ProbeTec CT/GC Q(x) assay, and 81.1% for the cobas CT/NG assay. Clinical sensitivities of the assays for N. gonorrhoeae, with limited positive results, ranged from 63.6% to 100%. Specificities for both infections ranged from 98.4 to 100%. Differences in analytical sensitivities and levels of molecular targets in clinical samples but not inhibitors of amplification may explain the differences in clinical sensitivities.

  2. Fluorescence detection in Lab-on-a-chip systems using ultrafast nucleic acid amplification methods

    NASA Astrophysics Data System (ADS)

    Gransee, Rainer; Schneider, Tristan; Elyorgun, Deniz; Strobach, Xenia; Schunck, Tobias; Gatscha, Theresia; Höth, Julian

    2014-05-01

    Today, nucleic amplification plays a key role in modern molecular biology allowing fast and specific laboratory diagnostics testing. An ultrafast microfluidic module (allowing 30 polymeric chain reaction (PCR) cycles in 6 minutes) based on an oscillating fluid plug concept was previously developed[1]. This system allows the amplification of native genomic deoxyribonucleic acid molecules (DNA) even from whole blood samples but still lacks some functionality compared to commercial bench top systems. This work presents the actual status of the renewed and advanced system, permitting the automated optical detection of not only the fluid plug position but also fluorescence detection. The system uses light emitting diodes (LED) for illumination and a low cost CMOS web-camera for optical detection. Image data processing allows the automated process control of the overall system components. Therefore, the system enables the performance of rapid and robust nucleic acid amplifications together with the integration of real time measurement technology. This allows the amplification and simultaneous quantification of the DNA molecules. The possibility to integrate swift nucleic amplification and optical detection into complex sample-to-answer analysis platforms opens up new pathways towards fast and transportable low-cost point of care devices.

  3. Instrument for Real-Time Digital Nucleic Acid Amplification on Custom Microfluidic Devices

    PubMed Central

    Selck, David A.

    2016-01-01

    Nucleic acid amplification tests that are coupled with a digital readout enable the absolute quantification of single molecules, even at ultralow concentrations. Digital methods are robust, versatile and compatible with many amplification chemistries including isothermal amplification, making them particularly invaluable to assays that require sensitive detection, such as the quantification of viral load in occult infections or detection of sparse amounts of DNA from forensic samples. A number of microfluidic platforms are being developed for carrying out digital amplification. However, the mechanistic investigation and optimization of digital assays has been limited by the lack of real-time kinetic information about which factors affect the digital efficiency and analytical sensitivity of a reaction. Commercially available instruments that are capable of tracking digital reactions in real-time are restricted to only a small number of device types and sample-preparation strategies. Thus, most researchers who wish to develop, study, or optimize digital assays rely on the rate of the amplification reaction when performed in a bulk experiment, which is now recognized as an unreliable predictor of digital efficiency. To expand our ability to study how digital reactions proceed in real-time and enable us to optimize both the digital efficiency and analytical sensitivity of digital assays, we built a custom large-format digital real-time amplification instrument that can accommodate a wide variety of devices, amplification chemistries and sample-handling conditions. Herein, we validate this instrument, we provide detailed schematics that will enable others to build their own custom instruments, and we include a complete custom software suite to collect and analyze the data retrieved from the instrument. We believe assay optimizations enabled by this instrument will improve the current limits of nucleic acid detection and quantification, improving our fundamental

  4. Instrument for Real-Time Digital Nucleic Acid Amplification on Custom Microfluidic Devices.

    PubMed

    Selck, David A; Ismagilov, Rustem F

    2016-01-01

    Nucleic acid amplification tests that are coupled with a digital readout enable the absolute quantification of single molecules, even at ultralow concentrations. Digital methods are robust, versatile and compatible with many amplification chemistries including isothermal amplification, making them particularly invaluable to assays that require sensitive detection, such as the quantification of viral load in occult infections or detection of sparse amounts of DNA from forensic samples. A number of microfluidic platforms are being developed for carrying out digital amplification. However, the mechanistic investigation and optimization of digital assays has been limited by the lack of real-time kinetic information about which factors affect the digital efficiency and analytical sensitivity of a reaction. Commercially available instruments that are capable of tracking digital reactions in real-time are restricted to only a small number of device types and sample-preparation strategies. Thus, most researchers who wish to develop, study, or optimize digital assays rely on the rate of the amplification reaction when performed in a bulk experiment, which is now recognized as an unreliable predictor of digital efficiency. To expand our ability to study how digital reactions proceed in real-time and enable us to optimize both the digital efficiency and analytical sensitivity of digital assays, we built a custom large-format digital real-time amplification instrument that can accommodate a wide variety of devices, amplification chemistries and sample-handling conditions. Herein, we validate this instrument, we provide detailed schematics that will enable others to build their own custom instruments, and we include a complete custom software suite to collect and analyze the data retrieved from the instrument. We believe assay optimizations enabled by this instrument will improve the current limits of nucleic acid detection and quantification, improving our fundamental

  5. Differential Item Functioning Amplification and Cancellation in a Reading Test

    ERIC Educational Resources Information Center

    Bao, Han; Dayton, C. Mitchell; Hendrickson, Amy B.

    2009-01-01

    When testlet effects and item idiosyncratic features are both considered to be the reasons of DIF in educational tests using testlets (Wainer & Kiely, 1987) or item bundles (Rosenbaum, 1988), it is interesting to investigate the phenomena of DIF amplification and cancellation due to the interactive effects of these two factors. This research…

  6. Instrument-free nucleic acid amplification assays for global health settings

    NASA Astrophysics Data System (ADS)

    LaBarre, Paul; Boyle, David; Hawkins, Kenneth; Weigl, Bernhard

    2011-06-01

    Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings.

  7. Instrument-free nucleic acid amplification assays for global health settings

    PubMed Central

    LaBarre, Paul; Boyle, David; Hawkins, Kenneth; Weigl, Bernhard

    2014-01-01

    Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings. PMID:25089171

  8. Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

    PubMed

    Kong, Janay E; Wei, Qingshan; Tseng, Derek; Zhang, Jingzi; Pan, Eric; Lewinski, Michael; Garner, Omai B; Ozcan, Aydogan; Di Carlo, Dino

    2017-03-02

    Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

  9. RNA internal standard synthesis by nucleic acid sequence-based amplification for competitive quantitative amplification reactions.

    PubMed

    Lo, Wan-Yu; Baeumner, Antje J

    2007-02-15

    Nucleic acid sequence-based amplification (NASBA) reactions have been demonstrated to successfully synthesize new sequences based on deletion and insertion reactions. Two RNA internal standards were synthesized for use in competitive amplification reactions in which quantitative analysis can be achieved by coamplifying the internal standard with the wild type sample. The sequences were created in two consecutive NASBA reactions using the E. coli clpB mRNA sequence as model analyte. The primer sequences of the wild type sequence were maintained, and a 20-nt-long segment inside the amplicon region was exchanged for a new segment of similar GC content and melting temperature. The new RNA sequence was thus amplifiable using the wild type primers and detectable via a new inserted sequence. In the first reaction, the forwarding primer and an additional 20-nt-long sequence was deleted and replaced by a new 20-nt-long sequence. In the second reaction, a forwarding primer containing as 5' overhang sequence the wild type primer sequence was used. The presence of pure internal standard was verified using electrochemiluminescence and RNA lateral-flow biosensor analysis. Additional sequence deletion in order to shorten the internal standard amplicons and thus generate higher detection signals was found not to be required. Finally, a competitive NASBA reaction between one internal standard and the wild type sequence was carried out proving its functionality. This new rapid construction method via NASBA provides advantages over the traditional techniques since it requires no traditional cloning procedures, no thermocyclers, and can be completed in less than 4 h.

  10. Methylmalonic acid blood test

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003565.htm Methylmalonic acid blood test To use the sharing features on this page, please enable JavaScript. The methylmalonic acid blood test measures the amount of methylmalonic acid ...

  11. Uric acid test (image)

    MedlinePlus

    Uric acid urine test is performed to check for the amount of uric acid in urine. Urine is collected over a 24 ... testing. The most common reason for measuring uric acid levels is in the diagnosis or treatment of ...

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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

  14. Stomach acid test

    MedlinePlus

    Gastric acid secretion test ... of the cells in the stomach to release acid. The stomach contents are then removed and analyzed. ... 3.5). These numbers are converted to actual acid production in units of milliequivalents per hour in ...

  15. Lactic acid test

    MedlinePlus

    Lactate test ... test. Exercise can cause a temporary increase in lactic acid levels. ... not getting enough oxygen. Conditions that can increase lactic acid levels include: Heart failure Liver disease Lung disease ...

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

    PubMed Central

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

    2015-01-01

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

  17. Nuclemeter: A Reaction-Diffusion Based Method for Quantifying Nucleic Acids Undergoing Enzymatic Amplification

    PubMed Central

    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

  18. Uric Acid Test

    MedlinePlus

    ... products and services. Advertising & Sponsorship: Policy | Opportunities Uric Acid Share this page: Was this page helpful? Also known as: Serum Urate; UA Formal name: Uric Acid Related tests: Synovial Fluid Analysis , Kidney Stone Analysis , ...

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

    PubMed

    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.

  20. Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing.

    PubMed

    Haines, Alicia M; Tobe, Shanan S; Kobus, Hilton J; Linacre, Adrian

    2015-10-01

    We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.

  1. Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

    PubMed Central

    Zanoli, Laura Maria; Spoto, Giuseppe

    2012-01-01

    Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed. PMID:25587397

  2. Powerful Amplification Cascades of FRET-Based Two-Layer Nonenzymatic Nucleic Acid Circuits.

    PubMed

    Quan, Ke; Huang, Jin; Yang, Xiaohai; Yang, Yanjing; Ying, Le; Wang, He; Xie, Nuli; Ou, Min; Wang, Kemin

    2016-06-07

    Nucleic acid circuits have played important roles in biological engineering and have increasingly attracted researchers' attention. They are primarily based on nucleic acid hybridizations and strand displacement reactions between nucleic acid probes of different lengths. Signal amplification schemes that do not rely on protein enzyme show great potential in analytical applications. While the single amplification circuit often achieves linear amplification that may not meet the need for detection of target in a very small amount, it is very necessary to construct cascade circuits that allow for larger amplification of inputs. Herein, we have successfully engineered powerful amplification cascades of FRET-based two-layer nonenzymatic nucleic acid circuits, in which the outputs of catalyzed hairpin assembly (CHA) activate hybridization chain reactions (HCR) circuits to induce repeated hybridization, allowing real-time monitoring of self-assembly process by FRET signal. The cascades can yield 50000-fold signal amplification with the help of the well-designed and high-quality nucleic acid circuit amplifiers. Subsequently, with coupling of structure-switching aptamer, as low as 200 pM adenosine is detected in buffer, as well as in human serum. To our knowledge, we have for the first time realized real-time monitoring adaptation of HCR to CHA circuits and achieved amplified detection of nucleic acids and small molecules with relatively high sensitivity.

  3. Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

    PubMed Central

    Abd El Galil, Khaled H.; El Sokkary, M. A.; Kheira, S. M.; Salazar, Andre M.; Yates, Marylynn V.; Chen, Wilfred; Mulchandani, Ashok

    2005-01-01

    A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water. PMID:16269748

  4. Visual detection of nucleic acids based on lateral flow biosensor and hybridization chain reaction amplification.

    PubMed

    Ying, Na; Ju, Chuanjing; Li, Zhongyi; Liu, Wensen; Wan, Jiayu

    2017-03-01

    In this study, a new lateral flow nucleic acid biosensor (LFNAB) using hybridization chain reaction (HCR) for signal amplification was developed for visual detection of nucleic acids with high sensitivity and low cost. A "sandwich-type" detection strategy was employed in our design. The sandwich system of capture probe (CP)/target DNA/reporter probe (RP)-HCR complexes was fabricated as the sensing platform. As the initiator strand, reporter probe propagated a chain reaction of hybridization events between the two hairpin probes modified with biotin, and determined whether long nicked DNA polymers were formed. The biotin-labeled double-strand DNA polymers then introduced numerous Streptavidin (SA)-labeled gold nanoparticles (AuNPs) on the lateral flow device. The CP/target DNA/RP-HCR complexes were captured on the test zone by the specific reaction between anti-Fam monoclonal antibody (anti-Fam mAb) on the test zone and Fam of the complexes. The accumulation of AuNPs on the test zone of the biosensor enabled the visual detection of specific sequences. The detection limit of specific DNA was as low as 1.76pM, which was about 2 orders lower than that of the LFNAB without HCR amplification. And the detection limit of Salmonella was 3×10(3)cfumL(-1). In conclusion, this visual detection system, HCR-LFNAB, is suitable for non-specialist personnel and point-of-care (POC) diagnosis in low-resource settings.

  5. Ternary surface monolayers for ultrasensitive (zeptomole) amperometric detection of nucleic acid hybridization without signal amplification.

    PubMed

    Wu, Jie; Campuzano, Susana; Halford, Colin; Haake, David A; Wang, Joseph

    2010-11-01

    A ternary surface monolayer, consisting of coassembled thiolated capture probe, mercaptohexanol and dithiothreitol, is shown to offer dramatic improvements in the signal-to-noise characteristics of electrochemical DNA hybridization biosensors based on common self-assembled monolayers. Remarkably low detection limits down to 40 zmol (in 4 μL samples) as well as only 1 CFU Escherichia coli per sensor are thus obtained without any additional amplification step in connection to the commonly used horseradish peroxidase/3,3',5,5'-tetramethylbenzidine system. Such dramatic improvements in the detection limits (compared to those of common binary alkanethiol interfaces and to those of most electrochemical DNA sensing strategies without target or signal amplification) are attributed primarily to the remarkably higher resistance to nonspecific adsorption. This reflects the highly compact layer (with lower pinhole density) produced by the coupling of the cyclic- and linear-configuration "backfillers" that leads to a remarkably low background noise even in the presence of complex sample matrixes. A wide range of surface compositions have been investigated, and the ternary mixed monolayer has been systematically optimized. Detailed impedance spectroscopy and cyclic voltammetric studies shed useful insights into the surface coverage. The impressive sensitivity and high specificity of the simple developed methodology indicate great promise for a wide range of nucleic acid testing, including clinical diagnostics, biothreat detection, food safety, and forensic analysis.

  6. One-step nucleic acid amplification (OSNA): where do we go with it?

    PubMed

    Tamaki, Yasuhiro

    2017-02-01

    The one-step nucleic acid amplification (OSNA) assay was initially developed for the intraoperative assessment of sentinel lymph node metastases in breast cancer. This assay measures cytokeratin 19 (CK19) mRNA copy number and is widely used in hospitals. The results of the IBCSG 23-01, ACOSOG Z0011, and AMAROS trials demonstrated that no further axillary dissection is required for patients with sentinel lymph nodes that tested positive for cancer, which has led to a decreasing trend in the need for intraoperative assessment of lymph nodes. Here, I review studies relevant to OSNA and discuss perspectives on future applications of OSNA in cancer surgery. The studies reviewed were identified by carrying out a search on PubMed for all articles pertaining to OSNA and published prior to the end of June 2016 using the keywords "OSNA" or "one-step nucleic acid amplification" in the title or abstract. Method comparison studies between OSNA and pathological assessment for the detection of lymph node metastasis in breast cancer revealed that in a pooled assessment OSNA had a high specificity (94.8 %), high concordant rate (93.8 %), and a negative predictive value (97.6 %). Similar results have been found for gastric, colorectal, and lung cancers in multicenter studies. These results demonstrate that OSNA can serve as an alternative method to pathological assessment for examining lymph node metastasis. Multicenter prospective studies with a large sample size are needed to definitively reveal the superiority of OSNA over pathological assessment to predict prognosis. Technical refinements to improve the assay are essential to its further development as a new standard for testing in place of pathological examination.

  7. An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids.

    PubMed

    Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R; Malamud, Daniel; Corstjens, Paul L A M; Bau, Haim H

    2010-08-01

    A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids.

  8. An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

    PubMed Central

    Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L. A. M.

    2010-01-01

    A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid—based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids. PMID:20401537

  9. Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification

    PubMed Central

    Shah, Kamal G.; Guelig, Dylan; Diesburg, Steven; Buser, Joshua; Burton, Robert; LaBarre, Paul; Richards-Kortum, Rebecca; Weigl, Bernhard

    2015-01-01

    Previous chemical heater designs for isothermal nucleic acid amplification have been based on solid-liquid phase transition, but using this approach, developers have identified design challenges en route to developing a low-cost, disposable device. Here, we demonstrate the feasibility of a new heater configuration suitable for isothermal amplification in which one reactant of an exothermic reaction is a liquid-gas phase-change material, thereby eliminating the need for a separate phase-change compartment. This design offers potentially enhanced performance and energy density compared to other chemical and electric heaters. PMID:26430883

  10. A Fully Integrated Paperfluidic Molecular Diagnostic Chip for the Extraction, Amplification, and Detection of Nucleic Acids from Clinical Samples

    PubMed Central

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

    2016-01-01

    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 onto 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 under 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. Multiplex-microsphere-quantitative polymerase chain reaction: nucleic acid amplification and detection on microspheres.

    PubMed

    Liang, Fang; Lai, Richard; Arora, Neetika; Zhang, Kang Liang; Yeh, Che-Cheng; Barnett, Graeme R; Voigt, Paul; Corrie, Simon R; Barnard, Ross T

    2013-01-01

    We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a "universal" oligonucleotide "tagged" polymerase chain reaction (PCR) forward primer, (ii) a sensor sequence complementary to the universal sequence on the forward PCR primer modified with a fluorescent dye, and (iii) a universal oligonucleotide coupled to Luminex microspheres. The PCR takes place with the microspheres present in the reaction tube. With the consumption of the universal oligonucleotide tagged forward primer, the fluorescently labeled sequences can bind to the universal oligonucleotide on the microspheres. We tested the microsphere quantitative PCR system with up to three different target genes (Neisseria meningitides porA and ctrA and influenza A M gene segment) as templates in a single PCR tube. The analytical sensitivity of this quantitative PCR system was tested and compared with the TaqMan system. The multiplex-microsphere-quantitative PCR system does not require design of unique internal probes for each target and has potential for a high degree of multiplexing, greater than the limited multiplexing achievable with current real-time protocols.

  12. Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

    NASA Technical Reports Server (NTRS)

    Fan, Wenhong (Inventor); Cassell, Alan M. (Inventor); Han, Jie (Inventor)

    2006-01-01

    Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

  13. Diagnosis of mycobacterial infections by nucleic acid amplification: 18-month prospective study.

    PubMed Central

    Kirschner, P; Rosenau, J; Springer, B; Teschner, K; Feldmann, K; Böttger, E C

    1996-01-01

    We have investigated the use of DNA amplification by PCR for the detection of mycobacteria in clinical specimens, with the gene encoding the 16S rRNA as a target. Following generic amplification of mycobacterial nucleic acids, screening was done with genus-specific probe; this was followed by species differentiation by use of highly discriminating probes or nucleic acid sequencing. In a prospective 18-month evaluation, criteria to select specimens for PCR analysis were defined. Of a total of 8,272 specimens received, 729 samples satisfied the criteria and were subjected to DNA amplification. Clinical specimens included material from the respiratory tract (sputa and bronchial washings), aspirates, biopsies, and various body fluids (cerebrospinal, pleural, peritoneal, and gastric fluids). After resolution of discrepant results, the sensitivity of the PCR assay was 84.5%, the specificity was 99.5%, the positive predictive value was 97.6%, and the negative predictive value was 96.4%. The sensitivity and negative predictive value of culture (with a combination of broth and solid media) were 77.5 and 94.8%, respectively. In conclusion, this PCR assay provides an efficient strategy to detect and identify multiple mycobacterial species and performs well in comparison with culture. PMID:8789005

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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. System for portable nucleic acid testing in low resource settings

    NASA Astrophysics Data System (ADS)

    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.

  17. Folic acid - test

    MedlinePlus

    ... folic acid before and during pregnancy helps prevent neural tube defects, such as spina bifida. Women who ... take more if they have a history of neural tube defects in earlier pregnancies. Ask your provider ...

  18. Citric acid urine test

    MedlinePlus

    ... used to diagnose renal tubular acidosis and evaluate kidney stone disease. Normal Results The normal range is 320 ... tubular acidosis and a tendency to form calcium kidney stones. The following may decrease urine citric acid levels: ...

  19. Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

    PubMed

    Sharma, Nidhi; Hoshika, Shuichi; Hutter, Daniel; Bradley, Kevin M; Benner, Steven A

    2014-10-13

    Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.

  20. An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification.

    PubMed

    Smith, Matthew C; Steimle, George; Ivanov, Stan; Holly, Mark; Fries, David P

    2007-08-29

    A compact hand-held heated fluorometric instrument for performing real-time isothermal nucleic acid amplification and detection is described. The optoelectronic instrument combines a Printed Circuit Board/Micro Electro Mechanical Systems (PCB/MEMS) reaction detection/chamber containing an integrated resistive heater with attached miniature LED light source and photo-detector and a disposable glass waveguide capillary to enable a mini-fluorometer. The fluorometer is fabricated and assembled in planar geometry, rolled into a tubular format and packaged with custom control electronics to form the hand-held reactor. Positive or negative results for each reaction are displayed to the user using an LED interface. Reaction data is stored in FLASH memory for retrieval via an in-built USB connection. Operating on one disposable 3 V lithium battery >12, 60 min reactions can be performed. Maximum dimensions of the system are 150 mm (h) x 48 mm (d) x 40 mm (w), the total instrument weight (with battery) is 140 g. The system produces comparable results to laboratory instrumentation when performing a real-time nucleic acid sequence-based amplification (NASBA) reaction, and also displayed comparable precision, accuracy and resolution to laboratory-based real-time nucleic acid amplification instrumentation. A good linear response (R2 = 0.948) to fluorescein gradients ranging from 0.5 to 10 microM was also obtained from the instrument indicating that it may be utilized for other fluorometric assays. This instrument enables an inexpensive, compact approach to in-field genetic screening, providing results comparable to laboratory equipment with rapid user feedback as to the status of the reaction.

  1. Rapid amplification/detection of nucleic acid targets utilizing a HDA/thin film biosensor.

    PubMed

    Jenison, Robert; Jaeckel, Heidi; Klonoski, Joshua; Latorra, David; Wiens, Jacinta

    2014-08-07

    Thin film biosensors exploit a flat, optically coated silicon-based surface whereupon formation of nucleic acid hybrids are enzymatically transduced in a molecular thin film that can be detected by the unaided human eye under white light. While the limit of sensitivity for detection of nucleic acid targets is at sub-attomole levels (60 000 copies) many clinical specimens containing bacterial pathogens have much lower levels of analyte present. Herein, we describe a platform, termed HDA/thin film biosensor, which performs helicase-dependant nucleic acid amplification on a thin film biosensor surface to improve the limit of sensitivity to 10 copies of the mecA gene present in methicillin-resistant strains of Staphylococcus. As double-stranded DNA is unwound by helicase it was either bound by solution-phase DNA primers to be copied by DNA polymerase or hybridized to surface immobilized probe on the thin film biosensor surface to be detected. Herein, we show that amplification reactions on the thin film biosensor are equivalent to in standard thin wall tubes, with detection at the limit of sensitivity of the assay occurring after 30 minutes of incubation time. Further we validate the approach by detecting the presence of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) from positive blood culture aliquots with high specificity (signal/noise ratio of 105).

  2. Visual, base-specific detection of nucleic acid hybridization using polymerization-based amplification.

    PubMed

    Hansen, Ryan R; Johnson, Leah M; Bowman, Christopher N

    2009-03-15

    Polymerization-based signal amplification offers sensitive visualization of biotinylated biomolecules functionalized to glass microarrays in a manner suitable for point-of-care use. Here we report using this method for visual detection of multiplexed nucleic acid hybridizations from complex media and develop an application toward point mutation detection and single nucleotide polymorphism (SNP) typing. Primer extension reactions were employed to label selectively and universally all complementary surface DNA hybrids with photoinitiators, permitting simultaneous and dynamic photopolymerization from positive sites to 0.5-nM target concentrations. Dramatic improvements in signal ratios between complementary and mismatched hybrids enabled visual discrimination of single base differences in KRAS codon-12 biomarkers.

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

    PubMed Central

    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 amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls. PMID:21602369

  4. Sensitive detection of nucleic acids with rolling circle amplification and surface-enhanced Raman scattering spectroscopy.

    PubMed

    Hu, Juan; Zhang, Chun-yang

    2010-11-01

    Detection of specific DNA sequences is important to molecular biology research and clinical diagnostics. To improve the sensitivity of surface-enhanced Raman scattering spectroscopy (SERS), a variety of signal amplification methods has been developed, including Raman-active-dye, polymerase chain reaction (PCR) technology, molecular beacon, SERS-active substrates, and SERS-tag. However, the combination of rolling circle amplification (RCA) with SERS for nucleic acid detection has not been reported. Herein, we describe a new approach for nucleic acid detection by the combination of RCA reaction with SERS. Because of the binding of abundance repeated sequences of RCA products with gold nanoparticle (Au NP) and Rox-modified detection probes, SERS signal is significantly amplified and the detection limit of 10.0 pM might be achieved. The sensitivity of RCA-based SERS has increased by as much as 3 orders of magnitude as compared to PCR-based SERS and is also comparable with or even exceeds that of both RCA-based electrochemical and RCA-based fluorescent methods. This RCA-based SERS might discriminate perfect matched target DNA from 1-base mismatched DNA with high selectivity. The high sensitivity and selectivity of RCA-based SERS makes it a potential tool for early diagnosis of gene-related disease and also offers a great promise for multiplexed assays with DNA microarrays.

  5. Systematic Evaluation of Different Nucleic Acid Amplification Assays for Cytomegalovirus Detection: Feasibility of Blood Donor Screening.

    PubMed

    Vollmer, T; Knabbe, C; Dreier, J

    2015-10-01

    Acute primary cytomegalovirus (CMV) infections, which commonly occur asymptomatically among blood donors, represent a significant risk for serious morbidity in immunocompromised patients (a major group of transfusion recipients). We implemented a routine CMV pool screening procedure for plasma for the identification of CMV DNA-positive donors, and we evaluated the sensitivities and performance of different CMV DNA amplification systems. Minipools (MPs) of samples from 18,405 individual donors (54,451 donations) were screened for CMV DNA using the RealStar CMV PCR assay (Altona Diagnostic Technologies), with a minimum detection limit of 11.14 IU/ml. DNA was extracted with a high-volume protocol (4.8 ml, Chemagic Viral 5K kit; PerkinElmer) for blood donor pool screening (MP-nucleic acid testing [NAT]) and with the Nuclisens easyMAG system (0.5 ml; bioMérieux) for individual donation (ID)-NAT. In total, six CMV DNA-positive donors (0.03%) were identified by routine CMV screening, with DNA concentrations ranging from 4.35 × 10(2) to 4.30 × 10(3) IU/ml. Five donors already showed seroconversion and detectable IgA, IgM, and/or IgG antibody titers (IgA(+)/IgM(+)/IgG(-) or IgA(+)/IgM(+)/IgG(+)), and one donor showed no CMV-specific antibodies. Comparison of three commercial assays, i.e., the RealStar CMV PCR kit, the Sentosa SA CMV quantitative PCR kit (Vela Diagnostics), and the CMV R-gene PCR kit (bioMérieux), for MP-NAT and ID-NAT showed comparably good analytical sensitivities, ranging from 10.23 to 11.14 IU/ml (MP-NAT) or from 37.66 to 57.94 IU/ml (ID-NAT). The clinical relevance of transfusion-associated CMV infections requires further investigation, and the evaluated methods present powerful basic tools providing sensitive possibilities for viral testing. The application of CMV MP-NAT facilitated the identification of one donor with a window-phase donation during acute primary CMV infection.

  6. A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes.

    PubMed

    Roy, Sharmili; Wei, Sim Xiao; Ying, Jean Liew Zhi; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

    2016-12-15

    Electrochemiluminescence (ECL) has been widely rendered for nucleic acid testing. Here, we integrate loop-mediated isothermal amplification (LAMP) with ECL technique for DNA detection and quantification. The target LAMP DNA bound electrostatically with [Ru(bpy)3](+2) on the carbon electrode surface, and an ECL reaction was triggered by tripropylamine (TPrA) to yield luminescence. We illustrated this method as a new and highly sensitive strategy for the detection of sequence-specific DNA from different meat species at picogram levels. The proposed strategy renders the signal amplification capacities of TPrA and combines LAMP with inherently high sensitivity of the ECL technique, to facilitate the detection of low quantities of DNA. By leveraging this technique, target DNA of Sus scrofa (pork) meat was detected as low as 1pg/µL (3.43×10(-1)copies/µL). In addition, the proposed technique was applied for detection of Bacillus subtilis DNA samples and detection limit of 10pg/µL (2.2×10(3)copies/µL) was achieved. The advantages of being isothermal, sensitive and robust with ability for multiplex detection of bio-analytes makes this method a facile and appealing sensing modality in hand-held devices to be used at the point-of-care (POC).

  7. Detection of human enteric viruses in oysters by in vivo and in vitro amplification of nucleic acids.

    PubMed Central

    Chung, H; Jaykus, L A; Sobsey, M D

    1996-01-01

    This study describes the detection of enteroviruses and hepatitis A virus in 31 naturally contaminated oyster specimens by nucleic acid amplification and oligonucleotide probing. Viruses were extracted by adsorption-elution-precipitation from 50-g oyster samples harvested from an area receiving sewage effluent discharge. Ninety percent of each extract was inoculated into primate kidney cell cultures for virus isolation and infectivity assay. Viruses in the remaining 10% of oyster extract that was not inoculated into cell cultures were further purified and concentrated by a procedure involving Freon extraction, polyethylene glycol precipitation, and Pro-Cipitate precipitation. After 3 to 4 weeks of incubation, RNA was extracted from inoculated cultures that were negative for cytopathic effects (CPE). These RNA extracts and the RNA from virions purified and concentrated directly from oyster extracts were subjected to reverse transcriptase PCR (RT-PCR) with primer pairs for human enteroviruses and hepatitis A virus. The resulting amplicons were confirmed by internal oligonucleotide probe hybridization. For the portions of oyster sample extracts inoculated into cell cultures, 12 (39%) were positive for human enteroviruses by CPE and 6 (19%) were positive by RT-PCR and oligoprobing of RNA extracts from CPE-negative cell cultures. For the remaining sample portions tested by direct RT-PCR and oligoprobing after further concentration, five (about 16%) were confirmed to be positive for human enteroviruses. Hepatitis A virus was also detected in RNA extracts of two CPE-positive samples by RT-PCR and oligoprobing. Combining the data from all three methods, enteric viruses were detected in 18 of 31 (58%) samples. Detection by nucleic acid methods increased the number of positive samples by 50% over detection by CPE in cell culture. Hence, nucleic acid amplification methods increase the detection of noncytopathic human enteric viruses in oysters. PMID:8837433

  8. Intraoperative diagnosis of sentinel lymph node metastases in breast cancer treatment with one-step nucleic acid amplification assay (OSNA)

    PubMed Central

    Szychta, Paweł; Westfal, Bogusław; Maciejczyk, Rafał; Smolarz, Beata; Romanowicz, Hanna; Krawczyk, Tomasz

    2016-01-01

    Introduction The aim of the study was to evaluate the clinical usefulness of a one-step nucleic acid amplification assay (OSNA) for intraoperative detection of metastases to sentinel lymph nodes (SLNs) in comparison to examination of frozen sections, and to summarize the results of previous studies. Material and methods We enrolled 98 patients aged 58.13 ±10.74 years treated surgically for breast cancer, and 99 biopsies of SLNs were followed by analysis of 105 SLNs. The central 1 mm slice of SLN was used for examination of frozen sections, whereas 2 outer slices of SLNs were analyzed intraoperatively with OSNA. Detection of isolated tumor cells (ITC), micrometastases or macrometastases with OSNA extended surgery to axillary lymph node dissection. Congruency of results was assessed between OSNA and examination of frozen sections. Results One-step nucleic acid amplification assay detected metastases in 29/105 SLNs in surgery of 27/99 breasts, including ITC in 3/29 SLNs, micrometastases in 12/29 and macrometastases in 14/29. One-step nucleic acid amplification assay detected significantly more metastases to SLNs than examination of frozen sections (p < 0.0001). All 8 inconsistent results were positive in OSNA and negative in examination of frozen sections; ITC were identified in 2/8 SLNs and micrometastases in 6/8 SLNs. Sensitivity for OSNA was calculated as 100%, specificity as 90.47%, and κ was 79.16%. Conclusions One-step nucleic acid amplification assay analysis allows rapid and quantitative detection of mRNA CK19 with high specificity and a low rate of false positives. One-step nucleic acid amplification assay is a reliable tool for intraoperative diagnosis of whole SLNs during surgery of breast cancer. One-step nucleic acid amplification assay minimizes the need for secondary surgery and avoids delays in the adjuvant treatment. PMID:27904514

  9. Analytical Performance and Clinical Utility of a Nucleic Acid Sequence-Based Amplification Assay for Detection of Cytomegalovirus Infection

    PubMed Central

    Witt, Donald J.; Kemper, M.; Stead, Andrew; Sillekens, P.; Ginocchio, Christine C.; Espy, Mark J.; Paya, Carlos V.; Smith, Thomas F.; Roeles, Frits; Caliendo, Angela M.

    2000-01-01

    A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection. PMID:11060058

  10. Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis - Lost in the Jungle of Methods, Targets, and Assays?

    PubMed

    Nolte, Oliver

    2012-01-01

    Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of 'pitfalls' is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and

  11. Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis – Lost in the Jungle of Methods, Targets, and Assays?

    PubMed Central

    Nolte, Oliver

    2012-01-01

    Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of ‘pitfalls’ is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and

  12. Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

    PubMed

    Shen, Feng; Davydova, Elena K; Du, Wenbin; Kreutz, Jason E; Piepenburg, Olaf; Ismagilov, Rustem F

    2011-05-01

    In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for "yes or no" digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37-42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader

  13. Enhanced nucleic acid amplification with blood in situ by wire-guided droplet manipulation (WDM)

    PubMed Central

    Harshman, Dustin K.; Reyes, Roberto; Park, Tu San; You, David J.; Song, Jae-Young; Yoon, Jeong-Yeol

    2013-01-01

    There are many challenges facing the use of molecular biology to provide pertinent information in a timely, cost effective manner. Wire-guided droplet manipulation (WDM) is an emerging format for conducting molecular biology with unique characteristics to address these challenges. To demonstrate the use of WDM, an apparatus was designed and assembled to automate polymerase chain reaction (PCR) on a reprogrammable platform. WDM minimizes thermal resistance by convective heat transfer to a constantly moving droplet in direct contact with heated silicone oil. PCR amplification of the GAPDH gene was demonstrated at a speed of 8.67 sec/cycle. Conventional PCR was shown to be inhibited by the presence of blood. WDM PCR utilizes molecular partitioning of nucleic acids and other PCR reagents from blood components, within the water-in-oil droplet, to increase PCR reaction efficiency with blood in situ. The ability to amplify nucleic acids in the presence of blood simplifies pre-treatment protocols towards true point-of-care diagnostic use. The 16s rRNA hypervariable regions V3 and V6 were amplified from Klebsiella pneumoniae genomic DNA with blood in situ. The detection limit of WDM PCR was 1 ng/µL or 105 genomes/µL with blood in situ. The application of WDM for rapid, automated detection of bacterial DNA from whole blood may have an enormous impact on the clinical diagnosis of infections in bloodstream or chronic wound/ulcer, and patient safety and morbidity. PMID:24140832

  14. Enhanced nucleic acid amplification with blood in situ by wire-guided droplet manipulation (WDM).

    PubMed

    Harshman, Dustin K; Reyes, Roberto; Park, Tu San; You, David J; Song, Jae-Young; Yoon, Jeong-Yeol

    2014-03-15

    There are many challenges facing the use of molecular biology to provide pertinent information in a timely, cost effective manner. Wire-guided droplet manipulation (WDM) is an emerging format for conducting molecular biology with unique characteristics to address these challenges. To demonstrate the use of WDM, an apparatus was designed and assembled to automate polymerase chain reaction (PCR) on a reprogrammable platform. WDM minimizes thermal resistance by convective heat transfer to a constantly moving droplet in direct contact with heated silicone oil. PCR amplification of the GAPDH gene was demonstrated at a speed of 8.67 s/cycle. Conventional PCR was shown to be inhibited by the presence of blood. WDM PCR utilizes molecular partitioning of nucleic acids and other PCR reagents from blood components, within the water-in-oil droplet, to increase PCR reaction efficiency with blood in situ. The ability to amplify nucleic acids in the presence of blood simplifies pre-treatment protocols towards true point-of-care diagnostic use. The 16s rRNA hypervariable regions V3 and V6 were amplified from Klebsiella pneumoniae genomic DNA with blood in situ. The detection limit of WDM PCR was 1 ng/μL or 10(5)genomes/μL with blood in situ. The application of WDM for rapid, automated detection of bacterial DNA from whole blood may have an enormous impact on the clinical diagnosis of infections in bloodstream or chronic wound/ulcer, and patient safety and morbidity.

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

    PubMed

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

    2011-08-25

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

  16. A colorimetric biosensor for detection of attomolar microRNA with a functional nucleic acid-based amplification machine.

    PubMed

    Li, Dandan; Cheng, Wei; Yan, Yurong; Zhang, Ye; Yin, Yibing; Ju, Huangxian; Ding, Shijia

    2016-01-01

    A functional nucleic acid-based amplification machine was designed for simple and label-free ultrasensitive colorimetric biosensing of microRNA (miRNA). The amplification machine was composed of a complex of trigger template and C-rich DNA modified molecular beacon (MB) and G-rich DNA (GDNA) as the probe, polymerase and nicking enzyme, and a dumbbell-shaped amplification template. The presence of target miRNA triggered MB mediated strand displacement to cyclically release nicking triggers, which led to a toehold initiated rolling circle amplification to produce large amounts of GDNAs. The formed GDNAs could stack with hemin to form G-quadruplex/hemin DNAzyme, a well-known horseradish peroxidase (HRP) mimic, for catalyzing a colorimetric reaction. The modified MB improved the stringent target recognition and reduced background signal. The proposed sensing strategy showed very high sensitivity and selectivity with a wide dynamic range from 10 aM to 1.0 nM, and enabled successful visual analysis of trace amount of miRNA in real sample by the naked eye. This rapid and highly efficient signal amplification strategy provided a simple and sensitive platform for miRNA detection. It would be a versatile and powerful tool for clinical molecular diagnostics.

  17. Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids.

    PubMed

    Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

    2016-01-01

    Absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification. However, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. Accordingly, isothermal methods, such as recombinase polymerase amplification (RPA), are more attractive. We developed a picoliter well array (PWA) chip with 27,000 consistently sized picoliter reactions (314 pL) for isothermal DNA quantification using digital RPA (dRPA) at 39°C. Sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., <20 μL). Passivating the chip surface using a methoxy-PEG-silane agent effectively eliminated cross-contamination during dRPA. Our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-cm(2) area. It was not necessary to use scan shooting and stitch serial small images together. Using this method, we quantified serial dilutions of a Listeria monocytogenes gDNA stock solution from 9 × 10(-1) to 4 × 10(-3) copies per well with an average error of less than 11% (N = 15). Overall dRPA-on-chip processing required less than 30 min, which was a 4-fold decrease compared to dPCR, requiring approximately 2 h. dRPA on the PWA chip provides a simple and highly sensitive method to quantify nucleic acids without thermal cycling or precise micropump/microvalve control. It has applications in fast field analysis and critical clinical diagnostics under resource-limited settings.

  18. Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids

    PubMed Central

    Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

    2016-01-01

    Absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification. However, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. Accordingly, isothermal methods, such as recombinase polymerase amplification (RPA), are more attractive. We developed a picoliter well array (PWA) chip with 27,000 consistently sized picoliter reactions (314 pL) for isothermal DNA quantification using digital RPA (dRPA) at 39°C. Sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., <20 μL). Passivating the chip surface using a methoxy-PEG-silane agent effectively eliminated cross-contamination during dRPA. Our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-cm2 area. It was not necessary to use scan shooting and stitch serial small images together. Using this method, we quantified serial dilutions of a Listeria monocytogenes gDNA stock solution from 9 × 10-1 to 4 × 10-3 copies per well with an average error of less than 11% (N = 15). Overall dRPA-on-chip processing required less than 30 min, which was a 4-fold decrease compared to dPCR, requiring approximately 2 h. dRPA on the PWA chip provides a simple and highly sensitive method to quantify nucleic acids without thermal cycling or precise micropump/microvalve control. It has applications in fast field analysis and critical clinical diagnostics under resource-limited settings. PMID:27074005

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

    PubMed

    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.

  20. Neighborhood disorder, sleep quality, and psychological distress: testing a model of structural amplification.

    PubMed

    Hill, Terrence D; Burdette, Amy M; Hale, Lauren

    2009-12-01

    Using data from the 2004 Survey of Texas Adults (n=1504), we examine the association between perceived neighborhood disorder and psychological distress. Building on previous research, we test whether the effect of neighborhood disorder is mediated and moderated by sleep quality. Our specific analytic strategy follows a two-stage theoretical model of structural amplification. In the first stage, perceptions of neighborhood disorder increase psychological distress indirectly by reducing sleep quality. In the second stage, the effect of neighborhood disorder on psychological distress is amplified by poor sleep quality. The results of our analyses are generally consistent with our theoretical model. We find that neighborhood disorder is associated with poorer sleep quality and greater psychological distress. We also observe that the positive association between neighborhood disorder and psychological distress is mediated (partially) and moderated (amplified) by poor sleep quality.

  1. Quantitative nucleic acid amplification methods and their implications in clinical virology

    PubMed Central

    Singh, Mini P; Galhotra, Shipra; Saigal, Karnika; Kumar, Archit; Ratho, Radha Kanta

    2017-01-01

    Recently, a number of techniques have been approved for quantification of viral nucleic acids in clinical samples. Viral load (VL) tests have considerable importance in the management of patients and are widely used in routine diagnosis. In clinical virology, VL testing are important to monitor the antiviral treatment, to initiate preemptive therapy, to understand pathogenesis, and to evaluate the infectivity. These tests have now become a part of many diagnostic and treatment guidelines. Considering the various challenges for in-house viral testing related to the standardization, validation, and precision; they are gradually being replaced by the United States Food and Drug Administration (US FDA) cleared tests. This review summarizes the various viral quantification methods and also discusses the clinical applicability of these in human immunodeficiency virus, Hepatitis B virus, Hepatitis C virus, Cytomegalovirus, and Epstein Barr virus infected patients. Further the challenges and future perspectives of VL testing have also been discussed. PMID:28251100

  2. Quantitative nucleic acid amplification methods and their implications in clinical virology.

    PubMed

    Singh, Mini P; Galhotra, Shipra; Saigal, Karnika; Kumar, Archit; Ratho, Radha Kanta

    2017-01-01

    Recently, a number of techniques have been approved for quantification of viral nucleic acids in clinical samples. Viral load (VL) tests have considerable importance in the management of patients and are widely used in routine diagnosis. In clinical virology, VL testing are important to monitor the antiviral treatment, to initiate preemptive therapy, to understand pathogenesis, and to evaluate the infectivity. These tests have now become a part of many diagnostic and treatment guidelines. Considering the various challenges for in-house viral testing related to the standardization, validation, and precision; they are gradually being replaced by the United States Food and Drug Administration (US FDA) cleared tests. This review summarizes the various viral quantification methods and also discusses the clinical applicability of these in human immunodeficiency virus, Hepatitis B virus, Hepatitis C virus, Cytomegalovirus, and Epstein Barr virus infected patients. Further the challenges and future perspectives of VL testing have also been discussed.

  3. A sensitive SERS assay for detecting proteins and nucleic acids using a triple-helix molecular switch for cascade signal amplification.

    PubMed

    Ye, Sujuan; Wu, Yanying; Zhang, Wen; Li, Na; Tang, Bo

    2014-08-25

    A novel surface-enhanced Raman scattering (SERS) detection system is developed for proteins and nucleic acids based on a triple-helix molecular switch for multiple cycle signal amplification, achieving high sensitivity, universality, rapid analysis, and high selectivity.

  4. Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis.

    PubMed

    Toley, Bhushan J; Covelli, Isabela; Belousov, Yevgeniy; Ramachandran, Sujatha; Kline, Enos; Scarr, Noah; Vermeulen, Nic; Mahoney, Walt; Lutz, Barry R; Yager, Paul

    2015-11-21

    We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in <20 minutes at 49 °C, which makes it one of the fastest existing isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible l-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in <30 minutes. We also present a simple kinetic model of iSDA that incorporates competition between target and primer-dimer amplification. This is the first model that quantitates the effects of primer-dimer products in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.

  5. Acid loading test (pH)

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003615.htm Acid loading test (pH) To use the sharing features on this page, please enable JavaScript. The acid loading test (pH) measures the ability of the ...

  6. A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

    PubMed Central

    LaBarre, Paul; Hawkins, Kenneth R.; Gerlach, Jay; Wilmoth, Jared; Beddoe, Andrew; Singleton, Jered; Boyle, David; Weigl, Bernhard

    2011-01-01

    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. PMID:21573065

  7. Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods.

    PubMed

    Rodríguez, Alicia; Werning, María L; Rodríguez, Mar; Bermúdez, Elena; Córdoba, Juan J

    2012-12-01

    A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain.

  8. Trichomonas Testing

    MedlinePlus

    ... Trichomonas vaginalis by Amplified Detection; Trichomonas vaginalis by Direct Fluorescent Antibody (DFA) Related tests: Pap Test , Chlamydia ... by one of the following methods: Molecular testing, direct DNA probes, or nucleic acid amplification tests (NAATs)— ...

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

    PubMed

    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 (<1 parasite μL(-1)) in a label-free and real-time manner. The developed system would be of great potential for better diagnosis of malaria in low-resource settings.

  10. Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor

    NASA Astrophysics Data System (ADS)

    Liu, Hongxing; Xing, Da; Zhou, Xiaoming

    2014-09-01

    Food-borne pathogens such as Listeria monocytogenes have been recognized as a major cause of human infections worldwide, leading to substantial health problems. Food-borne pathogen identification needs to be simpler, cheaper and more reliable than the current traditional methods. Here, we have constructed a low-cost paper biosensor for the detection of viable pathogenic bacteria with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper biosensor to perform a visual test, in which endpoint detection was performed using sandwich hybridization assays. When the RNA products migrated along the paper biosensor by capillary action, the gold nanoparticles accumulated at the designated Test line and Control line. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from Listeria monocytogenes could be detected. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. The developed method is suitable for point-of-care applications to detect food-borne pathogens, as it can effectively overcome the false-positive results caused by amplifying nonviable Listeria monocytogenes.

  11. Acid Tests and Basic Fun.

    ERIC Educational Resources Information Center

    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…

  12. 2-Ethylhexanoic Acid; Final Test Rule

    EPA Pesticide Factsheets

    EPA is issuing a final test rule, under section 4 of the Toxic Substances Control Act (TSCA), requiring manufacturers and processors of 2-ethylhexanoic acid (EHA, CAS No. 149-57-5) to conduct testing.

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

    PubMed Central

    Gulliksen, Anja; Keegan, Helen; Martin, Cara; O'Leary, John; Solli, Lars A.; Falang, Inger Marie; Grønn, Petter; Karlgård, Aina; Mielnik, Michal M.; Johansen, Ib-Rune; Tofteberg, Terje R.; Baier, Tobias; Gransee, Rainer; Drese, Klaus; Hansen-Hagge, Thomas; Riegger, Lutz; Koltay, Peter; Zengerle, Roland; Karlsen, Frank; Ausen, Dag; Furuberg, Liv

    2012-01-01

    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. PMID:22235204

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

    DOEpatents

    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.

  15. HIV Antibody Test

    MedlinePlus

    ... test is performed that detects the genetic material ( RNA ) of the virus. An HIV RNA test will detect HIV in most people by ... next test to perform is an HIV-1 RNA test (nucleic acid amplification test, NAAT). If the ...

  16. Detection of Vibrio cholerae by isothermal cross-priming amplification combined with nucleic acid detection strip analysis.

    PubMed

    Zhang, Xia; Du, Xin-Jun; Guan, Chun; Li, Ping; Zheng, Wen-Jie; Wang, Shuo

    2015-08-01

    Vibrio cholerae is a water- and food-borne human pathogen, and V. cholerae serotypes O1 and O139 have attracted attention because of their severe pathogenesis. However, non-O1, non-O139 cholera vibrios (NCVs) were also recently recognized as having virulence properties. In this study, we developed a cross-priming amplification (CPA) method for the detection of all serotypes of V. cholerae. The specificity of the CPA method was tested using a panel of 60 different bacterial strains. All of the V. cholerae strains showed positive results, and 41 other types of bacteria gave negative results. The limit of detection of the CPA method was 79.28 fg of genomic DNA, 4.2 × 10(2) CFU/ml for bacteria in pure culture, and 5.6 CFU per 25 g of sample with pre-enrichment. This method showed a higher sensitivity than the loop-mediated isothermal amplification (LAMP) method did and was more convenient to perform. These results indicate that the CPA method can be used for the rapid preliminary screening of V. cholerae.

  17. Yield and future issues of nucleic acid testing.

    PubMed

    Roth, W K; Seifried, E

    2001-06-01

    Despite the much lower actual yield than that estimated for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) nucleic acid testing (NAT)-only positives in the USA and Germany, look-back procedures have revealed that no HCV transmission has occurred in Germany since the introduction of NAT. This indicates sufficient sensitivity of the pool-PCR approach. The slow ramp-up of hepatitis B virus (HBV) however, may require a different approach. It has been shown in Germany that the pooling of samples followed by virus enrichment results in a significant yield. Single donation testing for HBV would not increase the yield, because virus enrichment from mini-pool results in a similar sensitivity to that of single donation testing. Both strategies may be useful for extending future NAT to HBV screening. New candidate viruses for NAT are Parvo B19 and hepatitis A virus (HAV) because of their extreme resistance to inactivation procedures. Their low pathogenicity and epidemiologic characteristics, however, make them candidate viruses only for pooled source plasma. The main future issues of NAT will be related to the automation of pooling, extraction and amplification as a single homogeneous process. Depending on the throughput, automated single donation NAT as demonstrated by the 'Tigris' system may be an option, as far as all transfusion-relevant viruses will be included. In the near future high throughput systems will rely on pooled donor samples, most probably in conjunction with efficient enrichment procedures. For these systems, automation of the extraction and amplification process will be one of the first steps. These procedures will also limitthe costs of NAT and keep it available for use with future candidate viruses.

  18. Detection of methicillin-resistant Staphylococcus aureus directly by loop-mediated isothermal amplification and direct cefoxitin disk diffusion tests.

    PubMed

    Metwally, L; Gomaa, N; Hassan, R

    2014-05-01

    We evaluated the utility of 2 methods for detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from signal-positive blood culture bottles: loop-mediated isothermal amplification (LAMP) assay, and direct cefoxitin disk diffusion (DCDD) test using a 30 μg cefoxitin disk. In parallel, standard microbiological identification and oxacillin susceptibility testing with MecA PCR was performed. Of 60 blood cultures positive for Gram-positive cocci in clusters, LAMP (via detection of the FemA and MecA genes) showed 100% sensitivity and specificity for identification of MRSA/MSSA. When coagulase-negative staphylococci were tested, sensitivity for detection of methicillin resistance was 91.7% and specificity was 100%. DCDD along with direct tube coagulase assay detected only 80.6% of MRSA/MSSA. LAMP showed higher diagnostic accuracy although DCDD was more cost-effective and did not require additional reagents or supplies.

  19. Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection.

    PubMed Central

    Guatelli, J C; Gingeras, T R; Richman, D D

    1989-01-01

    The enzymatic amplification of specific nucleic acid sequences in vitro has revolutionized the use of nucleic acid hybridization assays for viral detection. With this method, the copy number of a pathogen-specific sequence is increased several orders of magnitude before detection is attempted. The sensitivity and specificity of detection are thus markedly improved. Mullis and Faloona devised the first method of sequence amplification in vitro, the polymerase chain reaction (K.B. Mullis and F.A. Faloona, Methods Enzymol. 155:355-350, 1987). By this method, synthetic oligonucleotide primers direct repeated, target-specific, deoxyribonucleic acid-synthetic reactions, resulting in an exponential increase in the amount of the specific target sequence. The application of sequence amplification to viral detection was initially performed with human immunodeficiency virus type 1 and human T-cell lymphoma virus type I. In principle, however, this approach can be applied to the detection of any deoxyribonucleic or ribonucleic acid virus; the only requirement is that sufficient nucleotide sequence data exist to allow the synthesis of target-specific oligonucleotide primers. The use of target amplification in vitro will permit a variety of studies of viral pathogenesis which have not been feasible because of the low copy number of the viral nucleic acids in infected material. This approach is particularly applicable to the study of human retroviral infections, which are chronic and persistent and are characterized by low titers of virus in tissues. In addition, target amplification in vitro will facilitate the development of new methods of sequence detection, which will be useful for rapid viral diagnosis in the clinical laboratory. PMID:2650862

  20. Screening of organ and tissue donors for West Nile virus by nucleic acid amplification--a three year experience in Alberta.

    PubMed

    Tilley, Peter A G; Fox, Julie D; Lee, Bonita; Chui, Linda; Preiksaitis, Jutta

    2008-10-01

    West Nile Virus (WNV)-specific nucleic acid amplification testing (NAAT) of organ and tissue donors remains controversial. We report three years of WNV donor screening in Alberta Canada using NAAT. Between 2003 and 2005, 1549 initial specimens were received. A valid negative result was issued within the specified turnaround time on 1531 (98.8%). The initial NAAT was successful for 1393 samples (90%), while repeat testing using an alternate NAAT resolved a further 126 samples. For 12 of 14 donors, a second specimen provided a valid negative result. Failure to generate a valid negative result in time resulted in rescheduling of one living related organ transplant, and surgery proceeded in the absence of a final result in one multi-organ donation after risk assessment. For 11 tissue donors, tissues were discarded due to lack of a WNV result. Invalid results usually occurred on postmortem haemolyzed tissue donor samples due to inhibitory reactions. There were no confirmed positive donors, no false-positive results and no solid organs lost due to WNV testing. We conclude that WNV NAAT of organ and tissue donors can be implemented without compromising availability of donors but requires committed laboratory support.

  1. Sugar-assisted kinetic resolution of amino acids and amplification of enantiomeric excess of organic molecules.

    PubMed

    Córdova, Armando; Sundén, Henrik; Xu, Yongmei; Ibrahem, Ismail; Zou, Weibiao; Engqvist, Magnus

    2006-07-17

    The origins of biological homochirality have intrigued researchers since Pasteur's discovery of the optical activity of biomolecules. Herein, we propose and demonstrate a novel alternative for the evolution of homochirality that is not based on autocatalysis and forges a direct relationship between the chirality of sugars and amino acids. This process provides a mechanism in which a racemic mixture of an amino acid can catalyze the formation of an optically active organic molecule in the presence of a sugar product of low enantiomeric excess.

  2. Effect of metallic cations on the efficiency of DNA amplification. Implications for nucleic acid replication during early stages of life

    NASA Astrophysics Data System (ADS)

    Arribas, María; de Vicente, Aránzazu; Arias, Armando; Lázaro, Ester

    2005-04-01

    The process of catalysis of biochemical reactions has been essential since the first organic molecules appeared on Earth. As the complexity of the ensemble of primitive biomolecules was very low, primitive catalysts had necessarily to be very simple molecules or ions. The evolution of catalysts had to be in parallel with the evolution of the molecular species reacting. An example of this parallel evolution is nucleic acid polymerization. Synthesis of primitive short oligonucleotides could have been catalysed by metal ions either in solution or on the surface of minerals such as montmorillonite clays. Some oligonucleotides could start to function as templates for the synthesis of complementary copies and there is experimental evidence supporting the role also played by metal ions in this process. In later stages of evolution, a group of enzymatic proteins, nucleic acid polymerases, has been selected to catalyse nucleic acid replication. The presence of Mg2+ in the active centre of these enzymes suggests that evolution has preserved some of the primitive catalysts, including them as cofactors of more complex molecules. However, the reasons why Mg2+ was selected among other ions that possibly were present in primitive environments are unknown. In this paper we try to approach this question by analysing the amplification efficiency of the polymerase chain reaction of a DNA fragment in the presence of different metal ions. In some cases the conditions of the reaction have been displaced from optimum (by the presence of nucleotide imbalances and a suboptimal Mg2+concentration). The results obtained permit one to draw interesting conclusions about how some metallic cations can help replication to proceed in conditions of limited substrate availability, a circumstance that could have been frequent at prebiotic stages, when nucleic acid synthesis was dependent on the physico-chemical conditions of the environment.

  3. Loop-Mediated Isothermal Amplification Test for Trypanosoma gambiense Group 1 with Stem Primers: A Molecular Xenomonitoring Test for Sleeping Sickness

    PubMed Central

    Mburugu, Gitonga N.

    2017-01-01

    The World Health Organization has targeted Human African Trypanosomiasis (HAT) for elimination by 2020 with zero incidence by 2030. To achieve and sustain this goal, accurate and easy-to-deploy diagnostic tests for Gambian trypanosomiasis which accounts for over 98% of reported cases will play a crucial role. Most needed will be tools for surveillance of pathogen in vectors (xenomonitoring) since population screening tests are readily available. The development of new tests is expensive and takes a long time while incremental improvement of existing technologies that have potential for xenomonitoring may offer a shorter pathway to tools for HAT surveillance. We have investigated the effect of including a second set of reaction accelerating primers (stem primers) to the standard T. brucei gambiense LAMP test format. The new test format was analyzed with and without outer primers. Amplification was carried out using Rotorgene 6000 and the portable ESE Quant amplification unit capable of real-time data output. The stem LAMP formats indicated shorter time to results (~8 min), were 10–100-fold more sensitive, and indicated higher diagnostic sensitivity and accuracy compared to the standard LAMP test. It was possible to confirm the predicted product using ESE melt curves demonstrating the potential of combining LAMP and real-time technologies as possible tool for HAT molecular xenomonitoring. PMID:28321260

  4. fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor

    PubMed Central

    Huang, Guoliang; Huang, Qin; Ma, Li; Luo, Xianbo; Pang, Biao; Zhang, Zhixin; Wang, Ruliang; Zhang, Junqi; Li, Qi; Fu, Rongxin; Ye, Jiancheng

    2014-01-01

    A sensitive DNA isothermal amplification method for the detection of DNA at fM to aM concentrations for pathogen identification was developed using a non-stick-coated metal microfluidic bioreactor. A portable confocal optical detector was utilized to monitor the DNA amplification in micro- to nanoliter reaction assays in real-time, with fluorescence collection near the optical diffraction limit. The non-stick-coated metal microfluidic bioreactor, with a surface contact angle of 103°, was largely inert to bio-molecules, and DNA amplification could be performed in a minimum reaction volume of 40 nL. The isothermal nucleic acid amplification for Mycoplasma pneumoniae identification in the non-stick-coated microfluidic bioreactor could be performed at a minimum DNA template concentration of 1.3 aM, and a detection limit of three copies of genomic DNA was obtained. This microfluidic bioreactor offers a promising clinically relevant pathogen molecular diagnostic method via the amplification of targets from only a few copies of genomic DNA from a single bacterium. PMID:25475544

  5. 21 CFR 862.1450 - Lactic acid test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ....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 status... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactic acid test system. 862.1450 Section...

  6. 21 CFR 862.1450 - Lactic acid test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ....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 status... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lactic acid test system. 862.1450 Section...

  7. 21 CFR 862.1450 - Lactic acid test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ....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 status... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lactic acid test system. 862.1450 Section...

  8. 21 CFR 862.1450 - Lactic acid test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

  9. 21 CFR 862.1450 - Lactic acid test system.

    Code of Federal Regulations, 2010 CFR

    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...

  10. Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Simple and Rapid Detection of Promyelocytic Leukemia–Retinoic Acid Receptor α mRNA

    PubMed Central

    Hashimoto, Yuki; Hatayama, Yuki; Kojima, Nao; Morishita, Shota; Matsumoto, Satoko; Hosoda, Yuzuru; Hara, Ayako; Motokura, Toru

    2016-01-01

    Background Acute promyelocytic leukemia (APL) is a disease characterized by expression of Promyelocytic Leukemia–Retinoic Acid Receptor α (PML-RARα) chimeric mRNA. Although APL is curable, early death due to hemorrhage is a major problem. Here, we report the development of a simple and rapid diagnostic method for APL based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). Methods An RT-LAMP primer set was designed to detect three types of PML-RARα mRNA in a single reaction. Serial dilutions of plasmid DNA containing bcr1, bcr2, or bcr3 PML-RARα sequences and RNA extracted from bone marrow aspirates of 6 patients with APL were used to compare the results of RT-LAMP and nested PCR assays. Results Plasmid DNA was amplified by RT-LAMP, for which the reaction time was > 4 h shorter and the lower detection limit was higher than for nested RT-PCR. Six of 7 samples tested positive by both methods. Conclusion We developed an RT-LAMP assay for simple and rapid PML-RARα mRNA detection that may be clinically useful for point-of-care testing and APL diagnosis. PMID:28070163

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

    PubMed

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

    2013-03-01

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

  12. A sensitive and specific hyperbranched rolling circle amplification assay and test strip for white spot syndrome virus.

    PubMed

    Zhao, Yu-Ran; Yin, Wei-Li; Yue, Zhi-Qin; Li, Ba-Fang

    2014-01-01

    White spot syndrome virus (WSSV) is a global threat to the prawn industry, and there is no simple method for field-based testing of this virus. We designed a padlock probe and primers to the capsid protein gene VP28 of WSSV, and established a hyperbranched rolling circle amplification (HRCA) assay and a corresponding strip-based test. The assay and the test strip both had similar high accuracy and specificity, and their sensitivity was about 10 copies/μL, which is 100 times higher than conventional PCR. In this study, 68 batches of prawns were tested for WSSV with the HRCA assay and test strip, and the results were compared with the PCR assay. The results indicated that both the assay and test strip had accuracy similar to each other and to the PCR results. However, the assay and strip were more sensitive and user-friendly than PCR. Establishment of this method will provide a rapid detection of WSSV and also a basis for field-based detection of animal disease.

  13. A collaborative study to establish the 1st WHO International Standard for human cytomegalovirus for nucleic acid amplification technology.

    PubMed

    Fryer, Jacqueline F; Heath, Alan B; Minor, Philip D

    2016-07-01

    Variability in the performance of nucleic acid amplification technology (NAT)-based assays presents a significant problem in the diagnosis and management of human cytomegalovirus (HCMV) infections. Here we describe a collaborative study to evaluate the suitability of candidate reference materials to harmonize HCMV viral load measurements in a wide range of NAT assays. Candidate materials comprised lyophilized Merlin virus, liquid Merlin virus, liquid AD169 virus, and purified HCMV Merlin DNA cloned into a bacterial artificial chromosome. Variability in the laboratory mean HCMV concentrations determined for virus samples across the different assays was 2 log10. Variability for the purified DNA sample was higher (>3 log10). The agreement between laboratories was markedly improved when the potencies of the liquid virus samples were expressed relative to the lyophilized virus candidate. In contrast, the agreement between laboratories for the purified DNA sample was not improved. Results indicated the suitability of the lyophilized Merlin virus preparation as the 1st WHO International Standard for HCMV for NAT. It was established in October 2010, with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/162). It is intended to be used to calibrate secondary references, used in HCMV NAT assays, in IU.

  14. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

    PubMed

    Dmitrienko, E V; Khomiakova, E A; Pyshnaia; Bragin, A G; Vedernikov, V E; Pyshnyĭ, D V

    2010-01-01

    The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

  15. Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA).

    PubMed

    Lutz, Sascha; Weber, Patrick; Focke, Max; Faltin, Bernd; Hoffmann, Jochen; Müller, Claas; Mark, Daniel; Roth, Günter; Munday, Peter; Armes, Niall; Piepenburg, Olaf; Zengerle, Roland; von Stetten, Felix

    2010-04-07

    For the first time we demonstrate a self-sufficient lab-on-a-foil system for the fully automated analysis of nucleic acids which is based on the recently available isothermal recombinase polymerase amplification (RPA). The system consists of a novel, foil-based centrifugal microfluidic cartridge including prestored liquid and dry reagents, and a commercially available centrifugal analyzer for incubation at 37 degrees C and real-time fluorescence detection. The system was characterized with an assay for the detection of the antibiotic resistance gene mecA of Staphylococcus aureus. The limit of detection was <10 copies and time-to-result was <20 min. Microfluidic unit operations comprise storage and release of liquid reagents, reconstitution of lyophilized reagents, aliquoting the sample into < or = 30 independent reaction cavities, and mixing of reagents with the DNA samples. The foil-based cartridge was produced by blow-molding and sealed with a self-adhesive tape. The demonstrated system excels existing PCR based lab-on-a-chip platforms in terms of energy efficiency and time-to-result. Applications are suggested in the field of mobile point-of-care analysis, B-detection, or in combination with continuous monitoring systems.

  16. Clinical implications of nucleic acid amplification methods for the diagnosis of viral infections of the nervous system.

    PubMed

    Weber, T; Frye, S; Bodemer, M; Otto, M; Lüke, W

    1996-06-01

    Amplification of viral nucleic acids from the cerebrospinal fluid (CSF) has considerably improved the diagnosis of several acute, subacute and chronic viral infections of the nervous system. In herpes simplex virus (HSV) encephalitis (HSE) the polymerase chain reaction (PCR) has become the method of choice for the rapid, non invasive diagnosis. Other herpes virus associated diseases which can now be reliably diagnosed are encephalitis, ventriculoencephalitis, polymyeloradiculitis, myelitis and an inflammatory polyradiculoneuropathy caused by cytomegalovirus (CMV), HSV, varicella-zoster virus (VZV) or Epstein-Barr virus (EBV), EBV associated primary B-cell-lymphoma of the brain, acute aseptic meningitis in young adults allied with VZV, and meningoencephalitis with recurrent seizures due to human herpes virus type 6 (HHV-6). In AIDS patients, PCR has helped to differentiate lesions either due to the human immunodeficiency virus (HIV) itself or to opportunistic infections such as progressive multifocal leukoencephalopathy (PML) caused by JC virus (JCV) or CMV related complications. HIV can be detected early in the course of infection in the CSF and the amount of proviral DNA in CSF cells seems to be correlated with the severity and/or progression of neurological signs and symptoms. Acute epidemic aseptic meningitis caused by enterovirus infections can now be reliably diagnosed and typed by reverse transcriptase PCR (RT-PCR). Meningitis cases caused by vaccination with the Jeryl Lynn and Urabe vaccine strain of mumps virus have been identified using RT-PCR and sequencing of the amplified products (amplicon).

  17. 21 CFR 862.1295 - Folic acid test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Folic acid test system. 862.1295 Section 862.1295....1295 Folic acid test system. (a) Identification. A folic acid test system is a device intended to measure the vitamin folic acid in plasma and serum. Folic acid measurements are used in the diagnosis...

  18. 21 CFR 862.1290 - Fatty acids test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Fatty acids test system. 862.1290 Section 862.1290....1290 Fatty acids test system. (a) Identification. A fatty acids test system is a device intended to measure fatty acids in plasma and serum. Measurements of fatty acids are used in the diagnosis...

  19. 21 CFR 862.1295 - Folic acid test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Folic acid test system. 862.1295 Section 862.1295....1295 Folic acid test system. (a) Identification. A folic acid test system is a device intended to measure the vitamin folic acid in plasma and serum. Folic acid measurements are used in the diagnosis...

  20. 21 CFR 862.1290 - Fatty acids test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fatty acids test system. 862.1290 Section 862.1290....1290 Fatty acids test system. (a) Identification. A fatty acids test system is a device intended to measure fatty acids in plasma and serum. Measurements of fatty acids are used in the diagnosis...

  1. Development of real-time multiplex nucleic acid sequence-based amplification for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens.

    PubMed

    Loens, K; Beck, T; Ursi, D; Overdijk, M; Sillekens, P; Goossens, H; Ieven, M

    2008-01-01

    Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.

  2. Diagnostic test accuracy of loop-mediated isothermal amplification assay for Mycobacterium tuberculosis: systematic review and meta-analysis

    PubMed Central

    Nagai, Kenjiro; Horita, Nobuyuki; Yamamoto, Masaki; Tsukahara, Toshinori; Nagakura, Hideyuki; Tashiro, Ken; Shibata, Yuji; Watanabe, Hiroki; Nakashima, Kentaro; Ushio, Ryota; Ikeda, Misako; Narita, Atsuya; Kanai, Akinori; Sato, Takashi; Kaneko, Takeshi

    2016-01-01

    Diagnostic test accuracy of the loop-mediated isothermal amplification (LAMP) assay for culture proven tuberculosis is unclear. We searched electronic databases for both cohort and case-control studies that provided data to calculate sensitivity and specificity. The index test was any LAMP assay including both commercialized kits and in-house assays. Culture-proven M. tuberculosis was considered a positive reference test. We included 26 studies on 9330 sputum samples and one study on 315 extra-pulmonary specimens. For sputum samples, 26 studies yielded the summary estimates of sensitivity of 89.6% (95% CI 85.6–92.6%), specificity of 94.0% (95% CI 91.0–96.1%), and a diagnostic odds ratio of 145 (95% CI 93–226). Nine studies focusing on Loopamp MTBC yielded the summary estimates of sensitivity of 80.9% (95% CI 76.0–85.1%) and specificity of 96.5% (95% CI 94.7–97.7%). Loopamp MTBC had higher sensitivity and lower specificity for smear-positive sputa compared to smear-negative sputa. In-house assays showed higher sensitivity and lower specificity compared to Loopamp MTBC. LAMP promises to be a useful test for the diagnosis of TB, however there is still need to improve the assay to make it simpler, cheaper and more efficient to make it competitive against other PCR methods already available. PMID:27958360

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

    PubMed

    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.

  4. Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays.

    PubMed

    Mattiuzzo, Giada; Ashall, James; Doris, Kathryn S; MacLellan-Gibson, Kirsty; Nicolson, Carolyn; Wilkinson, Dianna E; Harvey, Ruth; Almond, Neil; Anderson, Robert; Efstathiou, Stacey; Minor, Philip D; Page, Mark

    2015-01-01

    The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA.

  5. Higher specificity of nucleic acid sequence-based amplification isothermal technology than of real-time PCR for quantification of HIV-1 RNA on dried blood spots.

    PubMed

    Mercier-Delarue, Severine; Vray, Muriel; Plantier, Jean Christophe; Maillard, Theodora; Adjout, Zidan; de Olivera, Fabienne; Schnepf, Nathalie; Maylin, Sarah; Simon, Francois; Delaugerre, Constance

    2014-01-01

    Dried blood spots (DBS) are widely proposed as a plasma surrogate for monitoring antiretroviral treatment efficacy based on the HIV-1 RNA level (viral load [VL]) in resource-limited settings. Interfering coamplification of cell-associated HIV-1 DNA during reverse transcription (RT)-PCR can be avoided by using nucleic acid sequence-based amplification (NASBA) technology, which is based on an RNA template and isothermic conditions. We analyzed VL values obtained with DBS and plasma samples by comparing isothermic NASBA (NucliSENS EasyQ HIV-1 V2.0; bioMérieux) with real-time RT-PCR (Cobas TaqMan HIV-1 V2.0; Roche). Samples from 197 HIV-1-infected patients were tested (non-B subtypes in 51% of the cases). Nucleic acid extractions were performed by use of NucliSENS EasyMAG (bioMérieux) and Cobas AmpliPrep (Roche) before the NASBA and RT-PCR quantifications, respectively. Both quantification assays have lower limits of detection of 20 (1.3) and 800 (2.9) log10 copies/ml (log) in plasma and DBS, respectively. The mean (DBS minus plasma) differences were -0.39 and -0.46 log, respectively, for RT-PCR and NASBA. RT-PCR on DBS identified virological failure in 122 of 126 patients (sensitivity, 97%) and viral suppression in 58 of 70 patients (specificity, 83%), yielding 12 false-positive results (median, 3.2 log). NASBA on DBS identified virological failure in 85 of 96 patients (sensitivity, 89%) and viral suppression in 95 of 97 patients (specificity, 98%) and yielded 2 false-positive results (3.0 log for both). Both technologies detected HIV-1 RNA in DBS at a threshold of 800 copies/ml. This higher specificity of NASBA technology could avoid overestimation of poor compliance or the emergence of resistance when monitoring antiretroviral efficacy with the DBS method.

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

    SciTech Connect

    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.

  7. 3q26 Amplification Is an Effective Negative Triage Test for LSIL: A Historical Prospective Study

    PubMed Central

    Heitmann, Erica R.; Lankachandra, Kamani M.; Wall, Jeff; Harris, George D.; McKinney, Hollie J.; Jalali, G. Reza; Verma, Yogita; Kershnar, Eric; Kilpatrick, Michael W.; Tsipouras, Petros; Harper, Diane M.

    2012-01-01

    Background Women with low grade squamous intraepithelial lesions (LSIL) at cervical cancer screening are currently referred for further diagnostic work up despite 80% having no precancerous lesion. The primary purpose of this study is to measure the test characteristics of 3q26 chromosome gain (3q26 gain) as a host marker of carcinogenesis in women with LSIL. A negative triage test may allow these women to be followed by cytology alone without immediate referral to colposcopy. Methods and Findings A historical prospective study was designed to measure 3q26 gain from the archived liquid cytology specimens diagnosed as LSIL among women attending colposcopy between 2007 and 2009. 3q26 gain was assessed on the index liquid sample; and sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were measured at immediate triage and at 6–16 months after colposcopic biopsy. The sensitivity of 3q26 gain measured at immediate triage from automated and manually reviewed tests in 65 non-pregnant unique women was 70% (95% CI: 35, 93) with a NPV of 89% (95% CI: 78, 96). The sensitivity and NPV increased to 80% (95% CI: 28, 99) and 98% (95% CI: 87, 100), respectively, when only the automated method of detecting 3q26 gain was used. Conclusions 3q26 gain demonstrates high sensitivity and NPV as a negative triage test for women with LSIL, allowing possible guideline changes to routine surveillance instead of immediate colposcopy. Prospective studies are ongoing to establish the sensitivity, specificity, PPV and NPV of 3q26 gain for LSIL over time. PMID:22792164

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

    PubMed

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

    2016-07-26

    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.

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

    PubMed Central

    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

  10. The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.

    PubMed

    Lewandowska, Marzena Anna; Czubak, Karol; Klonowska, Katarzyna; Jozwicki, Wojciech; Kowalewski, Janusz; Kozlowski, Piotr

    2015-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.

  11. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Pyruvic acid test system. 862.1655 Section 862....1655 Pyruvic acid test system. (a) Identification. A pyruvic acid test system is a device intended to measure pyruvic acid (an intermediate compound in the metabolism of carbohydrate) in plasma....

  12. 21 CFR 862.1795 - Vanilmandelic acid test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Vanilmandelic acid test system. 862.1795 Section... Systems § 862.1795 Vanilmandelic acid test system. (a) Identification. A vanilmandelic acid test system is a device intended to measure vanilmandelic acid in urine. Measurements of vanilmandelic...

  13. 21 CFR 862.1095 - Ascorbic acid test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Ascorbic acid test system. 862.1095 Section 862....1095 Ascorbic acid test system. (a) Identification. An ascorbic acid test system is a device intended to measure the level of ascorbic acid (vitamin C) in plasma, serum, and urine. Ascorbic...

  14. 21 CFR 862.1775 - Uric acid test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Uric acid test system. 862.1775 Section 862.1775....1775 Uric acid test system. (a) Identification. A uric acid test system is a device intended to measure uric acid in serum, plasma, and urine. Measurements obtained by this device are used in the...

  15. 21 CFR 862.1775 - Uric acid test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Uric acid test system. 862.1775 Section 862.1775....1775 Uric acid test system. (a) Identification. A uric acid test system is a device intended to measure uric acid in serum, plasma, and urine. Measurements obtained by this device are used in the...

  16. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Pyruvic acid test system. 862.1655 Section 862....1655 Pyruvic acid test system. (a) Identification. A pyruvic acid test system is a device intended to measure pyruvic acid (an intermediate compound in the metabolism of carbohydrate) in plasma....

  17. 21 CFR 862.1095 - Ascorbic acid test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Ascorbic acid test system. 862.1095 Section 862....1095 Ascorbic acid test system. (a) Identification. An ascorbic acid test system is a device intended to measure the level of ascorbic acid (vitamin C) in plasma, serum, and urine. Ascorbic...

  18. 21 CFR 862.1795 - Vanilmandelic acid test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Vanilmandelic acid test system. 862.1795 Section... Systems § 862.1795 Vanilmandelic acid test system. (a) Identification. A vanilmandelic acid test system is a device intended to measure vanilmandelic acid in urine. Measurements of vanilmandelic...

  19. Quantitative benedict test using bicinchoninic acid.

    PubMed

    Chen, Q; Klemm, N; Jeng, I M

    1989-10-01

    Cupric ion (Cu2+), in complex form, functions as a selective oxidizing agent for a variety of compounds in the qualitative Benedict test. Cupric ion is reduced to cuprous ion (Cu+) in the reaction. We found that the cuprous ion formed in this type of redox reaction could be detected and quantified using 2,2'-bicinchoninic acid. This reagent produced an intense purple complex with cuprous ion. The color development in this modified Benedict test was dependent on pH, temperature, and time. The reaction is insensitive to ethanol and sodium dodecyl sulfate. This improved method of the Benedict test has enhanced sensitivity and makes the quantitation of compounds possible. This method should be useful for studies of Benedict-positive compounds which are available only in small amounts.

  20. 21 CFR 862.1775 - Uric acid test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Uric acid test system. 862.1775 Section 862.1775...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1775 Uric acid test system. (a) Identification. A uric acid test system is a device intended to...

  1. 21 CFR 862.1775 - Uric acid test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Uric acid test system. 862.1775 Section 862.1775...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1775 Uric acid test system. (a) Identification. A uric acid test system is a device intended to...

  2. 21 CFR 862.1775 - Uric acid test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Uric acid test system. 862.1775 Section 862.1775...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1775 Uric acid test system. (a) Identification. A uric acid test system is a device intended to...

  3. 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.

    PubMed

    Verkooyen, Roel P; Noordhoek, Gerda T; Klapper, Paul E; Reid, Jim; Schirm, Jurjen; Cleator, Graham M; Ieven, Margareta; Hoddevik, Gunnar

    2003-07-01

    The 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 correctly reported, and of 506 weakly positive samples 466 (92.1%) were correctly reported. In 74 (72.5%) data sets correct results were reported on all samples, and 17 data sets (16.7%) showed either one false-negative or one false-positive result. In another 11 data sets, two or more incorrect results were reported, and two data sets reported a false-positive result on one negative sample. The Roche COBAS Amplicor test was performed in 44 (43%) data sets, the Abbott LCx assay was performed in 31 (30%) data sets, the Roche Amplicor manual assay was performed in 9 (9%) data sets, an in-house PCR was performed in 9 (9%) data sets, the Becton Dickinson ProbeTec ET assay was performed in 5 (4.9%) data sets, and the GenProbe TMA assay was performed in 4 (3.9%) data sets. The results of the Roche Amplicor manual (95.6% correct), COBAS Amplicor (97.0%), and Abbott LCx (94.8%) tests were comparable (P = 0.48). The results with the in-house PCR, BD ProbeTec ET, and GenProbe TMA tests were reported correctly in 88.6, 98, and 92.5% of the tests, respectively. Freeze-drying of clinical urine specimens proved to be a successful method for generating standardized, stable, and easy-to-transport samples for the detection of C. trachomatis by using NATs. Although the results, especially the specificity, for this proficiency panel were better than most quality control studies, sensitivity problems occurred frequently, underlining the need for good laboratory practice and reference reagents to monitor the

  4. 21 CFR 862.1509 - Methylmalonic acid (nonquantitative) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... methylmalonic acid (nonquantitative) test system is a device intended to identify methylmalonic acid in urine. The identification of methylmalonic acid in urine is used in the diagnosis and treatment...

  5. Establishment of the 1st World Health Organization International Standard for Plasmodium falciparum DNA for nucleic acid amplification technique (NAT)-based assays

    PubMed Central

    Padley, David J; Heath, Alan B; Sutherland, Colin; Chiodini, Peter L; Baylis, Sally A

    2008-01-01

    Background In order to harmonize results for the detection and quantification of Plasmodium falciparum DNA by nucleic acid amplification technique (NAT)-based assays, a World Health Organization (WHO) collaborative study was performed, evaluating a series of candidate standard preparations. Methods Fourteen laboratories from 10 different countries participated in the collaborative study. Four candidate preparations based upon blood samples parasitaemic for P. falciparum were evaluated in the study. Sample AA was lyophilized, whilst samples BB, CC and DD were liquid/frozen preparations. The candidate standards were tested by each laboratory at a range of dilutions in four independent assays, using both qualitative and quantitative NAT-based assays. The results were collated and analysed statistically. Results Twenty sets of data were returned from the participating laboratories and used to determine the mean P. falciparum DNA content for each sample. The mean log10 "equivalents"/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD. The freeze-dried preparation AA, was examined by accelerated thermal degradation studies and found to be highly stable. Conclusion On the basis of the collaborative study, the freeze-dried material, AA (NIBSC code No. 04/176) was established as the 1st WHO International Standard for P. falciparum DNA NAT-based assays and has been assigned a potency of 109 International Units (IU) per ml. Each vial contains 5 × 108 IU, equivalent to 0.5 ml of material after reconstitution. PMID:18652656

  6. Label-free and sensitive fluorescence detection of nucleic acid, based on combination of a graphene oxid /SYBR green I dye platform and polymerase assisted signal amplification

    NASA Astrophysics Data System (ADS)

    Zhu, Xiao; Xing, Da

    2012-12-01

    A new label-free isothermal fluorescence amplification detection for nucleic acid has been developed. In this paper, we first developed a novel sensitive and specific detection platform with an unmodified hairpin probe (HP) combination of the graphene oxid (GO)/ SYBR green I dye (SG), which was relied on the selective principle of adsorption and the high quenching efficiency of GO. Then for the application of this new strategy, we used Mirco RNA-21 (Mir-21) as the target to evaluate this working principle of our design. When the target was hybridizing with the HP and inducing its conformation of change, an efficient isothermal circular strand-displacement polymerization reaction was activating to assist the first signal amplification. In this format, the formed complex conformation of DNA would interact with its high affinity dye, then detached from the surface of GO after incubating with the platform of GO/intercalating dye. This reaction would accompany with obvious fluorescence recovery, and accomplish farther signal enhancement by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. By taking advantage of the multiple amplification of signal, this method exerted substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir-21 with attomole range. It is expected that this cost-effective GO based sensor might hold considerable potential to apply in bioanalysis studies.

  7. Sensitive electrochemical detection of telomerase activity using spherical nucleic acids gold nanoparticles triggered mimic-hybridization chain reaction enzyme-free dual signal amplification.

    PubMed

    Wang, Wen-Jing; Li, Jing-Jing; Rui, Kai; Gai, Pan-Pan; Zhang, Jian-Rong; Zhu, Jun-Jie

    2015-03-03

    We report an electrochemical sensor for telomerase activity detection based on spherical nucleic acids gold nanoparticles (SNAs AuNPs) triggered mimic-hybridization chain reaction (mimic-HCR) enzyme-free dual signal amplification. In the detection strategy, SNAs AuNPs and two hairpin probes were employed. SNAs AuNPs as the primary amplification element, not only hybridized with the telomeric repeats on the electrode to amplify signal but also initiated the subsequent secondary amplification, mimic-hybridization chain reaction of two hairpin probes. If the cells' extracts were positive for telomerase activity, SNAs AuNPs could be captured on the electrode. The carried initiators could trigger an alternative hybridization reaction of two hairpin probes that yielded nicked double helices. The signal was further amplified enzyme-free by numerous hexaammineruthenium(III) chloride ([Ru(NH3)6](3+), RuHex) inserting into double-helix DNA long chain by electrostatic interaction, each of which could generate an electrochemical signal at appropriate potential. With this method, a detection limit of down to 2 HeLa cells and a dynamic range of 10-10,000 cells were achieved. Telomerase activities of different cell lines were also successfully evaluated.

  8. Detection of Campylobacter jejuni and Campylobacter coli in chicken meat samples by real-time nucleic acid sequence-based amplification with molecular beacons.

    PubMed

    Churruca, E; Girbau, C; Martínez, I; Mateo, E; Alonso, R; Fernández-Astorga, A

    2007-06-10

    A nucleic acid sequence-based amplification (NASBA) assay based on molecular beacons was used for real-time detection of Campylobacter jejuni and Campylobacter coli in samples of chicken meat. A set of specific primers and beacon probe were designed to target the 16S rRNA of both species. The real-time NASBA protocol including the RNA isolation was valid for both of the cell suspensions in buffered saline and the artificially contaminated chicken meat samples. The presence of rRNA could be correlated with cellular viability, following inactivation of the bacteria by heating, in inoculated chicken meat samples but not in RNase-free cell suspensions.

  9. Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

    PubMed

    Xu, Yao; Zheng, Zhi

    2016-05-15

    We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate.

  10. Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease—Towards a Point-of-Care Test

    PubMed Central

    Beissner, Marcus; Phillips, Richard Odame; Battke, Florian; Bauer, Malkin; Badziklou, Kossi; Sarfo, Fred Stephen; Maman, Issaka; Rhomberg, Agata; Piten, Ebekalisai; Frimpong, Michael; Huber, Kristina Lydia; Symank, Dominik; Jansson, Moritz; Wiedemann, Franz Xaver; Banla Kere, Abiba; Herbinger, Karl-Heinz; Löscher, Thomas; Bretzel, Gisela

    2015-01-01

    Background As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. Methodology/Principal Findings Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. Conclusions/Significance Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold

  11. Simplified diagnosis of malaria infection: GFM/PCR/ELISA a simplified nucleic acid amplification technique by PCR/ELISA.

    PubMed

    Machado, R L; Garret, D O; Adagu, I S; Warhurst, D C; Póvoa, M M

    1998-01-01

    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.

  12. A label-free signal amplification assay for DNA detection based on exonuclease III and nucleic acid dye SYBR Green I.

    PubMed

    Zheng, Aihua; Luo, Ming; Xiang, Dongshan; Xiang, Xia; Ji, Xinghu; He, Zhike

    2013-09-30

    We have developed a new fluorescence method for specific single-stranded DNA sequences with exonuclease III (Exo III) and nucleic acid dye SYBR Green I. It is demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 27 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of the target DNA, the hairpin-probe is in the stem-closed structure, the fluorescence of SYBR Green I is very strong. In the presence of the target DNA, the hairpin-probe hybridizes with the target DNA to form double-stranded structure with a blunt 3'-terminus. Thus, in the presence of Exo III, only the 3'-terminus of probe is subjected to digestion. Exo III catalyzes the stepwise removal of mononucleotides from this terminus, releasing the target DNA. The released target DNA then hybridizes with another probe, whence the cycle starts anew. The signal of SYBR Green I decreases greatly. This system provides a detection limit of 160 pM, which is comparable to the existing signal amplification methods that utilized Exo III as a signal amplification nuclease. Due to the unique property of Exo III, this method shows excellent detection selectivity for single-base discrimination. More importantly, superiors to other methods based on Exo III, these probes have the advantages of easier to design, synthesize, purify and thus are much cheaper and more applicable. This new approach could be widely applied to sensitive and selective nucleic acids detection.

  13. 30 CFR 7.48 - Acid resistance test.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Acid resistance test. 7.48 Section 7.48 Mineral... MINING PRODUCTS TESTING BY APPLICANT OR THIRD PARTY Battery Assemblies § 7.48 Acid resistance test. (a... solution of sulfuric acid (H2 SO4) by mixing 853 ml of water with 199 ml of sulfuric acid (H2 SO4) with...

  14. Hearing Loss and Cognitive-Communication Test Performance of Long-Term Care Residents With Dementia: Effects of Amplification

    ERIC Educational Resources Information Center

    Hopper, Tammy; Slaughter, Susan E.; Hodgetts, Bill; Ostevik, Amberley; Ickert, Carla

    2016-01-01

    Purpose: The study aims were (a) to explore the relationship between hearing loss and cognitive-communication performance of individuals with dementia, and (b) to determine if hearing loss is accurately identified by long-term care (LTC) staff. The research questions were (a) What is the effect of amplification on cognitive-communication test…

  15. An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care.

    PubMed

    Choi, Jane Ru; Hu, Jie; Tang, Ruihua; Gong, Yan; Feng, Shangsheng; Ren, Hui; Wen, Ting; Li, XiuJun; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

    2016-02-07

    With advances in point-of-care testing (POCT), lateral flow assays (LFAs) have been explored for nucleic acid detection. However, biological samples generally contain complex compositions and low amounts of target nucleic acids, and currently require laborious off-chip nucleic acid extraction and amplification processes (e.g., tube-based extraction and polymerase chain reaction (PCR)) prior to detection. To the best of our knowledge, even though the integration of DNA extraction and amplification into a paper-based biosensor has been reported, a combination of LFA with the aforementioned steps for simple colorimetric readout has not yet been demonstrated. Here, we demonstrate for the first time an integrated paper-based biosensor incorporating nucleic acid extraction, amplification and visual detection or quantification using a smartphone. A handheld battery-powered heating device was specially developed for nucleic acid amplification in POC settings, which is coupled with this simple assay for rapid target detection. The biosensor can successfully detect Escherichia coli (as a model analyte) in spiked drinking water, milk, blood, and spinach with a detection limit of as low as 10-1000 CFU mL(-1), and Streptococcus pneumonia in clinical blood samples, highlighting its potential use in medical diagnostics, food safety analysis and environmental monitoring. As compared to the lengthy conventional assay, which requires more than 5 hours for the entire sample-to-answer process, it takes about 1 hour for our integrated biosensor. The integrated biosensor holds great potential for detection of various target analytes for wide applications in the near future.

  16. 30 CFR 7.48 - Acid resistance test.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Acid resistance test. 7.48 Section 7.48 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR TESTING, EVALUATION, AND APPROVAL OF MINING PRODUCTS TESTING BY APPLICANT OR THIRD PARTY Battery Assemblies § 7.48 Acid resistance test....

  17. 30 CFR 7.48 - Acid resistance test.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Acid resistance test. 7.48 Section 7.48 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR TESTING, EVALUATION, AND APPROVAL OF MINING PRODUCTS TESTING BY APPLICANT OR THIRD PARTY Battery Assemblies § 7.48 Acid resistance test....

  18. 30 CFR 7.48 - Acid resistance test.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Acid resistance test. 7.48 Section 7.48 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR TESTING, EVALUATION, AND APPROVAL OF MINING PRODUCTS TESTING BY APPLICANT OR THIRD PARTY Battery Assemblies § 7.48 Acid resistance test....

  19. Biomaterials in light amplification

    NASA Astrophysics Data System (ADS)

    Mysliwiec, Jaroslaw; Cyprych, Konrad; Sznitko, Lech; Miniewicz, Andrzej

    2017-03-01

    Biologically produced or inspired materials can serve as optical gain media, i.e. they can exhibit the phenomenon of light amplification. Some of these materials, under suitable dye-doping and optical pumping conditions, show lasing phenomena. The emerging branch of research focused on obtaining lasing action in highly disordered and highly light scattering materials, i.e. research on random lasing, is perfectly suited for biological materials. The use of biomaterials in light amplification has been extensively reported in the literature. In this review we attempt to report on progress in the development of biologically derived systems able to show the phenomena of light amplification and random lasing together with the contribution of our group to this field. The rich world of biopolymers modified with molecular aggregates and nanocrystals, and self-organized at the nanoscale, offers a multitude of possibilities for tailoring luminescent and light scattering properties that are not easily replicated in conventional organic or inorganic materials. Of particular importance and interest are light amplification and lasing, or random lasing studies in biological cells and tissues. In this review we will describe nucleic acids and their complexes employed as gain media due to their favorable optical properties and ease of manipulation. We will report on research conducted on various biomaterials showing structural analogy to nucleic acids such as fluorescent proteins, gelatins in which the first distributed feedback laser was realized, and also amyloids or silks, which, due to their dye-doped fiber-like structure, allow for light amplification. Other materials that were investigated in that respect include polysaccharides, like starch exhibiting favorable photostability in comparison to other biomaterials, and chitosan, which forms photonic crystals or cellulose. Light amplification and random lasing was not only observed in processed biomaterials but also in living

  20. Enzymatic amplification-free nucleic acid hybridisation sensing on nanostructured thick-film electrodes by using covalently attached methylene blue.

    PubMed

    García-González, Raquel; Costa-García, Agustín; Fernández-Abedul, M Teresa

    2015-09-01

    Amplification-free (referring to enzymatic amplification step) detection methodologies are increasing in biosensor development due to the need of faster and simpler protocols. However, for maintaining sensitivity without this step, highly detectable molecules or very sensitive detection techniques are required. The nanostructuration of transducer surfaces with carbon nanotubes (CNTs), gold nanoparticles (AuNPs) or both in nanohybrid configurations has been employed in this work for DNA hybridisation sensing purposes. Methylene blue (MB), covalently attached to single stranded DNA, (ssDNA) was incubated with a complementary sequence immobilized on nanostructured screen-printed electrodes (AuSPEs). Although CNTs can increase notoriously the signal of the marker, adsorptive properties should also be considered when bioassays are performed because non-specific adsorption (NSA) phenomena are magnified. In this work, strategies for decreasing NSA were thoroughly evaluated for the detection of Mycoplasma pneumoniae (MP) on CNTs-nanostructured screen-printed electrodes. Among them, the employ of UV-radiation or long incubation times (72h) allowed obtaining higher signals for the complementary strand with respect to the non-complementary one. The use of CNTs/AuNPs nanohybrids, together with the use of streptavidin-biotin (ST-B) interaction allows the higher differentiation (with a 3.5 ratio) in the genosensing of M. pneumoniae.

  1. Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica.

    PubMed

    Kubota, Ryo; Labarre, Paul; Weigl, Bernhard H; Li, Yong; Haydock, Paul; Jenkins, Daniel M

    2013-04-01

    We report on the use of a novel non-instrumented platform to enable a Loop Mediated isothermal Amplification (LAMP) based assay for Salmonella enterica. Heat energy is provided by addition of a small amount (<150 g) of boiling water, and the reaction temperature is regulated by storing latent energy at the melting temperature of a lipid-based engineered phase change material. Endpoint classification of the reaction is achieved without opening the reaction tube by observing the fluorescence of sequence-specific FRET-based assimilating probes with a simple handheld fluorometer. At or above 22°C ambient temperature the non-instrumented devices could maintain reactions above a threshold temperature of 61°C for over 90 min-significantly longer than the 60 min reaction time. Using the simple format, detection limits were less than 20 genome copies for reactions run at ambient temperatures ranging from 8 to 36°C. When used with a pre-enrichment step and non-instrumented DNA extraction device, trace contaminations of Salmonella in milk close to 1 CFU/mL could be reliably detected. These findings illustrate that the non- instrumented amplification approach is a simple, viable, low-cost alternative for field-based food and agricultural diagnostics or clinical applications in developing countries.

  2. A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules.

    PubMed

    Mayr, Reinhard; Haider, Michaela; Thünauer, Roland; Haselgrübler, Thomas; Schütz, Gerhard J; Sonnleitner, Alois; Hesse, Jan

    2016-04-15

    Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single-stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra-sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39 fM (831 molecules in 35.4 nl assay volume) for target DNA and 16 fM (338 molecules) for target RNA after 1h on-chip hybridization.

  3. Detection of trace amounts of target DNA from massive background of nucleic acids by using LM-PCR-based pre-amplification method.

    PubMed

    Pan, Xiaoming; Wang, Jing; Zhang, Yanfang; Dong, Ping; Li, Chunchuan; Liang, Xingguo

    2016-11-08

    The sensitivity and specificity of DNA detection may decrease when the target DNA is in very low abundance. To effectively detect trace amounts of target DNA from massive background of nucleic acids, we have developed a powerful multiplex pre-amplification method based on ligation-mediated PCR (LM-PCR) that can greatly enrich multiple target DNAs from massive backgrounds. By employing type IIS restriction endonuclease (REase) and specifically designed oligonucleotide adapters, target DNA can be pre-amplified with high efficiency and sensitivity. Combining with normal PCR, ten copies of target DNA was effectively detected from over 10(8) times more excessive backgrounds with high specificity and ten times more effectively than conventional PCR. In particular, the usage of universal primer in the pre-amplification PCR (pre-amp PCR) ensured that multiple targets could be equivalently amplified, which was confirmed by quantitative PCR (qPCR), indicating it could meet the demands of high-throughput detection. The flexibility and applicability of pre-amp PCR was validated by using different microorganisms DNA as targets and employing two different type IIS REases. The results suggest that the pre-amp PCR method has broad application prospects in various gene detection fields. This article is protected by copyright. All rights reserved.

  4. Visual detection and differentiation of Classic Swine Fever Virus strains using nucleic acid sequence-based amplification (NASBA) and G-quadruplex DNAzyme assay

    PubMed Central

    Lu, Xiaolu; Shi, Xueyao; Wu, Gege; Wu, Tiantian; Qin, Rui; Wang, Yi

    2017-01-01

    The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses. PMID:28287135

  5. 21 CFR 862.1509 - Methylmalonic acid (nonquantitative) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Methylmalonic acid (nonquantitative) test system. 862.1509 Section 862.1509 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Test Systems § 862.1509 Methylmalonic acid (nonquantitative) test system. (a) Identification....

  6. Acid Test For Annealing Of Welds

    NASA Technical Reports Server (NTRS)

    Deese, Gary E.; Ellgass, Joseph P.

    1989-01-01

    Solution changes color if heat-treated condition lost. Simple test indicates whether welded joint retained its postweld heat-treated condition after reworking including rewelding and grinding. Test used instead of Rockwell or Brinell hardness tests when reworked surface inaccessible to hardness-testing apparatus or when small surface imperfections created by apparatus unacceptable.

  7. A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds.

    PubMed Central

    Pérez-Llarena, F J; Liras, P; Rodríguez-García, A; Martín, J F

    1997-01-01

    A regulatory gene (ccaR), located within the cephamycin gene cluster of Streptomyces clavuligerus, is linked to a gene (blp) encoding a protein similar to a beta-lactamase-inhibitory protein. Expression of ccaR is required for cephamycin and clavulanic acid biosynthesis in S. clavuligerus. The ccaR-encoded protein resembles the ActII-ORF4, RedD, AfsR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs. Disruption of ccaR by targeted double recombination resulted in the loss of the ability to synthesize cephamycin and clavulanic acid. Complementation of the disrupted mutant with ccaR restored production of both secondary metabolites. ccaR was expressed as a monocistronic transcript at 24 and 48 h in S. clavuligerus cultures (preceding the phase of antibiotic accumulation), but no transcript hybridization signals were observed at 72 or 96 h. This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis. Amplification of ccaR in S. clavuligerus resulted in a two- to threefold increase in the production of cephamycin and clavulanic acid. PMID:9068654

  8. Isothermal Multiple Displacement Amplification

    PubMed Central

    Luthra, Rajyalakshmi; Medeiros, L. Jeffrey

    2004-01-01

    Isothermal multiple strand displacement amplification (IMDA) of the whole human genome is a promising method for procuring abundant DNA from valuable and often limited clinical specimens. However, whether DNA generated by this method is of high quality and a faithful replication of the DNA in the original specimen, allowing for subsequent molecular diagnostic testing, requires verification. In this study, we evaluated the suitability of IMDA-generated DNA (IMDA-DNA) for detecting antigen receptor gene rearrangements, chromosomal translocations, and gene mutations using Southern blot analysis, polymerase chain reaction (PCR) methods, or sequencing methods in 28 lymphoma and leukemia clinical specimens. Molecular testing before and after whole genome amplification of these specimens using the IMDA technique showed concordance in 27 of 28 (96%) specimens. Analysis of IMDA-DNA by Southern blot analysis detected restriction fragments >12 kilobases long. No amplification bias was observed at all loci tested demonstrating that this method can be useful in generating large amounts of unbiased, high molecular weight DNA from limited clinical specimens. PMID:15269301

  9. Capture and Direct Amplification of DNA on Chitosan Microparticles in a Single PCR-Optimal Solution.

    PubMed

    Pandit, Kunal R; Nanayakkara, Imaly A; Cao, Weidong; Raghavan, Srinivasa R; White, Ian M

    2015-11-03

    While nucleic acid amplification tests have great potential as tools for rapid diagnostics, complicated sample preparation requirements inhibit their use in near-patient diagnostics and low-resource-setting applications. Recent advancements in nucleic acid purification have leveraged pH-modulated charge switching polymers to reduce the number of steps required for sample preparation. The polycation chitosan (pKa 6.4) has been used to efficiently purify DNA by binding nucleic acids in acidic buffers and then eluting them at a pH higher than 8.0. Though it is an improvement over conventional methods, this multistep procedure has not transformed the application of nucleic acid amplification assays. Here we describe a simpler approach using magnetic chitosan microparticles that interact with DNA in a manner that has not been reported before. The microparticles capture DNA at a pH optimal for PCR (8.5) just as efficiently as at low pH. Importantly, the captured DNA is still accessible by polymerase, enabling direct amplification from the microparticles. We demonstrate quantitative PCR from DNA captured on the microparticles, thus eliminating nearly all of the sample preparation steps. We anticipate that this new streamlined method for preparing DNA for amplification will greatly expand the diagnostic applications of nucleic acid amplification tests.

  10. Monte Carlo Modeling-Based Digital Loop-Mediated Isothermal Amplification on a Spiral Chip for Absolute Quantification of Nucleic Acids.

    PubMed

    Xia, Yun; Yan, Shuangqian; Zhang, Xian; Ma, Peng; Du, Wei; Feng, Xiaojun; Liu, Bi-Feng

    2017-03-21

    Digital loop-mediated isothermal amplification (dLAMP) is an attractive approach for absolute quantification of nucleic acids with high sensitivity and selectivity. Theoretical and numerical analysis of dLAMP provides necessary guidance for the design and analysis of dLAMP devices. In this work, a mathematical model was proposed on the basis of the Monte Carlo method and the theories of Poisson statistics and chemometrics. To examine the established model, we fabricated a spiral chip with 1200 uniform and discrete reaction chambers (9.6 nL) for absolute quantification of pathogenic DNA samples by dLAMP. Under the optimized conditions, dLAMP analysis on the spiral chip realized quantification of nucleic acids spanning over 4 orders of magnitude in concentration with sensitivity as low as 8.7 × 10(-2) copies/μL in 40 min. The experimental results were consistent with the proposed mathematical model, which could provide useful guideline for future development of dLAMP devices.

  11. A double signal amplification platform for ultrasensitive and simultaneous detection of ascorbic acid, dopamine, uric acid and acetaminophen based on a nanocomposite of ferrocene thiolate stabilized Fe₃O₄@Au nanoparticles with graphene sheet.

    PubMed

    Liu, Meiling; Chen, Qiong; Lai, Cailang; Zhang, Youyu; Deng, Jianhui; Li, Haitao; Yao, Shouzhuo

    2013-10-15

    A double signal amplification platform for ultrasensitive and simultaneous detection of ascorbic acid (AA), dopamine (DA), uric acid (UA) and acetaminophen (AC) was fabricated by a nanocomposite of ferrocene thiolate stabilized Fe₃O₄@Au nanoparticles with graphene sheet. The platform was constructed by coating a newly synthesized phenylethynyl ferrocene thiolate (Fc-SAc) modified Fe₃O₄@Au NPs coupling with graphene sheet/chitosan (GS-chitosan) on a glassy carbon electrode (GCE) surface. The Fe₃O₄@Au-S-Fc/GS-chitosan modified GCE exhibits a synergistic catalytic and amplification effect toward AA, DA, UA and AC oxidation. The oxidation peak currents of the four compounds on the electrode were linearly dependent on AA, DA, UA and AC concentrations in the ranges of 4-400 μM, 0.5-50 μM, 1-300 μM and 0.3-250 μM in the individual detection of each component, respectively. By simultaneously changing the concentrations of AA, DA, UA and AC, their electrochemical oxidation peaks appeared at -0.03, 0.15, 0.24 and 0.35 V, and good linear current responses were obtained in the concentration ranges of 6-350, 0.5-50, 1-90 and 0.4-32 μM with the detection limits of 1, 0.1, 0.2 and 0.05 μM (S/N=3), respectively.

  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.

    PubMed

    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.

  13. Testing Consent Order for Acrylic Acid

    EPA Pesticide Factsheets

    This document announces that EPA has signed an enforceable testing Consent Order with BASF Corporation, Dow Chemical U.S.A., Hoechst Celanese Chemical Group, Rohm and Haas Company, and Union Carbide Chemicals and Plastics, Inc.

  14. Evaluation of loop-mediated isothermal amplification for detection of Toxoplasma gondii in water samples and comparative findings by polymerase chain reaction and immunofluorescence test (IFT).

    PubMed

    Sotiriadou, Isaia; Karanis, Panagiotis

    2008-12-01

    The development and evaluation of a 1-step single-tube accelerated loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Toxoplasma in water samples is described. The method has been evaluated based on the amplification of B1 and TgOWP Toxoplasma genes, and it demonstrated a sensitivity detection limit of 0.1 tachyzoites' DNA for both genes. LAMP detection was evaluated and compared with nested polymerase chain reaction (PCR) in 26 water sample pellets spiked with known numbers of Toxoplasma oocysts. After DNA extraction, the detection sensitivity in spiked pellets was 100% by LAMP and 53.8% by PCR. Subsequently, 52 natural water samples of different origin were directly investigated by 3 assays: LAMP, PCR, and immunofluorescence test (IFT). Twenty-five (48%) of 52 have been found positive for Toxoplasma DNA by LAMP, whereas nested PCR products were generated in 7 of 52 (13.5%) water samples. All 52 water samples were negative for Toxoplasma by IFT. These data clearly indicate LAMP as a rapid, specific, and sensitive tool for the detection of Toxoplasma contamination in water samples.

  15. 30 CFR 7.48 - Acid resistance test.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... MINING PRODUCTS TESTING BY APPLICANT OR THIRD PARTY Battery Assemblies § 7.48 Acid resistance test. (a) Test procedures. (1) Prepare one sample each of the insulated surfaces of the battery box and of the... insulation plus the battery cover or box material. The insulation thickness shall be representative of...

  16. Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar

    PubMed Central

    Morris, Ulrika; Ding, Xavier C.; Jovel, Irina; Msellem, Mwinyi I.; Bergman, Daniel; Islam, Atiqul; Ali, Abdullah S.; Polley, Spencer; Gonzalez, Iveth J.; Mårtensson, Andreas; Björkman, Anders

    2017-01-01

    Background New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. Results The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3–2.4) and 0.7% (95%CI 0.4–1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0–55.8) and the specificity was 99.9% (CI95% 99.8–100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2–770) and HTP-LAMP negative (1.4 p/μL, range 0.1–7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Conclusions Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination. PMID:28095434

  17. Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

    PubMed Central

    2010-01-01

    Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum PMID:21143837

  18. Comparative detection of rotavirus RNA by conventional RT-PCR, TaqMan RT-PCR and real-time nucleic acid sequence-based amplification.

    PubMed

    Mo, Qiu-Hua; Wang, Hai-Bo; Tan, Hua; Wu, Bi-Mei; Feng, Zi-Li; Wang, Qi; Lin, Ji-Can; Yang, Ze

    2015-03-01

    Rotavirus is one of the major viral pathogens leading to diarrhea. Diagnosis has been conducted by either traditional cultural, serological methods or molecular biology techniques, which include RT-PCR and nucleic acid sequence-based amplification (NASBA). However, their differences regarding accuracy and sensitivity remain unknown. In this study, an in-house conventional RT-PCR assay and more importantly, an in-house real-time NASBA (RT-NASBA) were established, and compared with a commercial TaqMan RT-PCR assay. The results showed that all of these methods were able to detect and distinguish rotavirus from other diarrhea viruses with a 100% concordance rate during the course of an evaluation on 20 clinical stool samples. However, RT-NASBA was much quicker than the other two methods. More importantly, the limit of detection of RT-NASBA could reach seven copies per reaction and was one to two logs lower than that of conventional RT-PCR and TaqMan RT-PCR. These results indicate that this in-house assay was more sensitive, and thus could be used as an efficient diagnosis tool for rotavirus. To the best of our knowledge, this is the first direct comparison among three different assays for the detection of rotavirus. These findings would provide implication for the rational selection of diagnosis tool for rotavirus.

  19. Comparison and evaluation of real-time PCR, real-time nucleic acid sequence-based amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae.

    PubMed

    Templeton, Kate E; Scheltinga, Sitha A; Graffelman, A Willy; Van Schie, Jolanda M; Crielaard, Jantine W; Sillekens, Peter; Van Den Broek, Peterhans J; Goossens, Herman; Beersma, Matthias F C; Claas, Eric C J

    2003-09-01

    Mycoplasma pneumoniae is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of M. pneumoniae in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with Mycoplasma-positive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of M. pneumoniae, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.

  20. Detection of novel swine origin influenza A virus (H1N1) by real-time nucleic acid sequence-based amplification.

    PubMed

    Ge, Yiyue; Cui, Lunbiao; Qi, Xian; Shan, Jun; Shan, Yunfeng; Qi, Yuhua; Wu, Bing; Wang, Hua; Shi, Zhiyang

    2010-02-01

    Rapid detection of novel swine origin influenza A virus (S-OIV) (H1N1) is crucial for timely implementation of infection control measures. In this study, a haemagglutinin (HA) gene-based real-time nucleic acid sequence-based amplification (NASBA) assay was developed for the specific detection of S-OIV (H1N1). The assay was evaluated and validated by comparing it with existing detection methods for S-OIV (H1N1). Results obtained in a 10-fold dilution series assay demonstrated the analytic sensitivity of the present assay was comparable to that of a commercial S-OIV (H1N1) real-time RT-PCR kit and higher than that of the Centers for Disease Control and Prevention (CDC) TaqMan assay. The actual detection limit of the real-time NASBA assay was approximately 50 copies per reaction. Compared with reference methods (viral culture, conventional RT-PCR, and real-time RT-PCR), the sensitivity, specificity, positive predictive value, and negative predictive value of the present assay were all 100%. Overall, the results showed that the real-time NASBA assay could be used for sensitive and specific detection of S-OIV (H1N1).

  1. Selective adsorption and chiral amplification of amino acids in vermiculite clay-implications for the origin of biochirality.

    PubMed

    Fraser, Donald G; Fitz, Daniel; Jakschitz, T; Rode, Bernd M

    2011-01-21

    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-NH(3)Cl 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 NH(3)Cl 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 organic molecules in clay inter-layers is known to produce Layer-by-Layer or Langmuir-Blodgett films. Moreover atomistic simulations show that self-organization of organic species in clay interlayers is important. These non-centrosymmetric, chirally active nanofilms may cause clays to act subsequently as chiral amplifiers, concentrating organic material from dilute solution and having different adsorption energetics for D- and L-enantiomers. The additional role of clays in RNA oligomerization already postulated by Ferris and others, together with the need for the organization of amphiphilic molecules and lipids noted by Szostak and others, suggests that such chiral separation by clays in lagoonal environments at normal biological temperatures might also have played a significant role in the origin of biochirality.

  2. Test-Retest Reliability of 10 Hz Conditioning Electrical Stimulation Inducing Long-Term Potentiation (LTP)-Like Pain Amplification in Humans

    PubMed Central

    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 future crossover and parallel study designs. Methods The 10 Hz paradigm (500 rectangular pulses lasting 50 s) was repeated on two separate days with one week interval in twenty volunteers. Perceptual intensities to single electrical stimulation (SES) at the conditioned skin site and to mechanical stimuli (pinprick and light stroking) in immediate vicinity to the conditioned skin site were recorded. Superficial blood flow (SBF) was assessed as indicator of neurogenic inflammation. All outcome measures were assessed with 10 min interval three times before and six times after the CES. The coefficient of variation and intra-class correlation coefficient were calculated within session and between sessions. Sample sizes were estimated for future crossover (Ncr) and parallel (Np) drug testing studies expected to detect a 30% decrease for the individual outcome measure following 10 Hz CES. Results Perceptual intensity ratings to light stroking (Ncr = 2, Np = 33) and pinprick stimulation (491 mN) (Ncr = 6, Np = 54) increased after CES and showed better reliability in crossover than parallel design. The SBF increased after CES, and then declined until reaching a plateau 20 minutes postCES. SBF showed 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. PMID:27529175

  3. [Principle of LAMP method--a simple and rapid gene amplification method].

    PubMed

    Ushikubo, Hiroshi

    2004-06-01

    So far nucleic acid test (NAT) has been employed in various fields, including infectious disease diagnoses. However, due to its complicated procedures and relatively high cost, it has not been widely utilized in many actual diagnostic applications. We have therefore developed a simple and rapid gene amplification technology, Loop-mediated Isothermal Amplification (LAMP) method, which has shown prominent results of surpassing the performance of the conventional gene amplification methods. LAMP method acquires three main features: (1) all reaction can be carried out under isothermal conditions; (2) the amplification efficiency is extremely high and tremendous amount of amplification products can be obtained; and (3) the reaction is highly specific. Furthermore, developed from the standard LAMP method, a rapid LAMP method, by adding in the loop primers, can reduce the amplification time from the previous 1 hour to less than 30 minutes. Enormous amount of white precipitate of magnesium pyrophosphate is produced as a by-product of the amplification, therefore, direct visual detection is possible without using any reaction indicators and detection equipments. We believe LAMP technology, with the integration of these features, can rightly apply to clinical genetic testing, food and environmental analysis, as well as NAT in different fields.

  4. Investigations of ascorbic acid interference in urine test strips.

    PubMed

    Nagel, Dietmar; Seiler, Dieter; Hohenberger, Ewald F; Ziegler, Manfred

    2006-01-01

    Ascorbic acid at higher concentration in urine samples can lead to false negative results in a number of urine tests, with a potential risk of clinical findings being overlooked, particularly with glucose and hemoglobin. For this reason, the ascorbic acid status of urine samples should always be routinely known so as to establish what adjustment needs to be made. A much better approach, however, is to use a test which is by design largely resistant to ascorbic acid. We compared five very common 10-parameter urine test strips from different manufacturers. The results of this study show that of the strips tested, only the product Combur-Test from Roche Diagnostics is largely resistant to ascorbic acid interference. Even lowest - but clinically relevant - concentrations of erythrocytes (10/microL), hemoglobin (0.03 mg/dL), and glucose (50 mg/dL) were correctly detected with concentrations of up to 400 mg/L ascorbic acid. Higher analyte concentrations correctly reacted positive even in the presence of up to 1000 mg/L ascorbic acid.

  5. AFB (Acid-Fast Bacillus) Smear and Culture

    MedlinePlus

    ... Fast Bacillus Smear and Culture and Sensitivity; Mycobacteria tuberculosis Nucleic Acid Amplification Test Related tests: TB Screening ... is most commonly used to identify an active tuberculosis (TB) infection caused by the most medically important ...

  6. Development and implementation of real-time nucleic acid amplification for the detection of enterovirus infections in comparison to rapid culture of various clinical specimens.

    PubMed

    van Doornum, G J J; Schutten, M; Voermans, J; Guldemeester, G J J; Niesters, H G M

    2007-12-01

    Several real-time PCR and nucleic acid sequence-based amplification (NASBA) primer pairs and a modified real-time PCR primer pair for the detection of enteroviruses were compared. The modified real-time PCR primer pair was evaluated on clinical samples in comparison with cell culture using the MagnaPure LC Isolation instrument for nucleic acid extraction. Six hundred forty samples could be examined both by cell culture and real-time PCR. Faecal specimens (n = 285), cerebrospinal fluid (n = 210), throat swabs (n = 113), biopsies (n = 1--, vesicular fluid (n = 11), and pleural fluid specimens (n = 9) were included. By culture, 26/640 (4%) samples were positive for enterovirus. By real-time PCR, the number of positive specimens was 50 (7.8%). Of the 210 cerebrospinal fluid samples, three were positive by culture and nine by real-time PCR. Seventeen and 33 of a total of 285 faecal specimens were positive by culture and real-time PCR, respectively. In case of discrepant results, the clinical symptoms were in accordance with an infection due to enteroviruses. Genotyping using the VP1 gene correlated with serotyping by neutralization. In contrast, six of the 19 specimens that could be typed both by neutralization and by sequencing using the VP4 domain yielded a different genotype, yet within the same species. Real-time PCR turned out to be suitable for the detection of enteroviruses in the daily routine setting. In comparison to rapid culture, it offers a rapid, more sensitive, and reliable assay; especially in cerebrospinal fluid, the yield of enteroviruses is much higher.

  7. Amplification of electrolyte uptake in the absorptive glass mat (AGM) separator for valve regulated lead acid (VRLA) batteries

    NASA Astrophysics Data System (ADS)

    Kumar, Vijay; Kameswara Rao, P. V.; Rawal, Amit

    2017-02-01

    Absorptive glass mat (AGM) separators are widely used for valve regulated lead acid (VRLA) batteries due to their remarkable fiber and structural characteristics. Discharge performance and recharge effectiveness of VRLA batteries essentially rely on the distribution and saturation levels of the electrolyte within the AGM separator. Herein, we report an analytical model for predicting the wicking characteristics of AGM battery separators under unconfined and confined states. The model of wicking behavior of AGM is based upon Fries and Dreyer's approach that included the effect of gravity component which was neglected in classic Lucas-Washburn's model. In addition, the predictive model of wicking accounted for realistic structural characteristics of AGM via orientation averaging approach. For wicking under confined state, the structural parameters have been updated under defined level of compressive stresses based upon the constitutive equation derived for a planar network of fibers in AGM under transverse loading conditions. A comparison has been made between the theoretical models and experimental results of wicking behavior under unconfined and confined states. Most importantly, the presented work has highlighted the questionable validity of classic Lucas-Washburn model for predicting the wicking characteristics of AGM separator over longer time duration.

  8. Chemical amplification based on fluid partitioning

    DOEpatents

    Anderson, Brian L.; Colston, Jr., Billy W.; Elkin, Chris

    2006-05-09

    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.

  9. Charge Efficiency Tests of Lead/Acid Batteries

    NASA Technical Reports Server (NTRS)

    Rowlette, J. J.

    1984-01-01

    Current, voltage, and gas evolution measured during charge/discharge cycles. Series of standarized tests for evaluating charging efficiency of lead/acid storage batteries described in report. Purpose of tests to provide information for design of battery charger that allows maximum recharge efficiency for electric-vehicle batteries consistent with other operating parameters, such as range, water loss, and cycle life.

  10. 21 CFR 862.1320 - Gastric acidity test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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 §...

  11. 21 CFR 862.1295 - Folic acid test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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 §...

  12. 21 CFR 862.1295 - Folic acid test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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 §...

  13. 21 CFR 862.1290 - Fatty acids test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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 §...

  14. 21 CFR 862.1290 - Fatty acids test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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 §...

  15. 21 CFR 862.1295 - Folic acid test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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 §...

  16. 21 CFR 862.1095 - Ascorbic acid test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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 §...

  17. 21 CFR 862.1290 - Fatty acids test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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 §...

  18. 21 CFR 862.1095 - Ascorbic acid test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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 §...

  19. 21 CFR 862.1095 - Ascorbic acid test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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 §...

  20. 21 CFR 862.1320 - Gastric acidity test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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 862.1320 - Gastric acidity test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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 §...

  2. 21 CFR 862.1320 - Gastric acidity test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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 §...

  3. 21 CFR 862.1320 - Gastric acidity test system.

    Code of Federal Regulations, 2010 CFR

    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 §...

  4. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Pyruvic acid test system. 862.1655 Section 862.1655 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 §...

  5. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Pyruvic acid test system. 862.1655 Section 862.1655 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 §...

  6. Internal control for nucleic acid testing based on the use of purified Bacillus atrophaeus subsp. globigii spores.

    PubMed

    Picard, François J; Gagnon, Martin; Bernier, Marthe R; Parham, Nicholas J; Bastien, Martine; Boissinot, Maurice; Peytavi, Régis; Bergeron, Michel G

    2009-03-01

    Commonly used internal controls (ICs) to monitor the efficiency of nucleic acid testing (NAT) assays do not allow verification of nucleic acid extraction efficiency. Since microbial cells are often difficult to lyse, it is important to ensure that nucleic acids are efficiently extracted from any target organism. For this purpose, we developed a cellular IC based on the use of nonpathogenic Bacillus spores. Purified Bacillus atrophaeus subsp. globigii (referred to hereafter as simply B. atrophaeus) spores were added to vaginal and anal samples, which were then subjected to rapid DNA extraction and subsequent PCR amplification. The proof of concept of this cellular IC was made through the use of both manual and automated DNA extraction methods, using vaginal or anal samples spiked with B. atrophaeus spores, combined with a multiplex real-time PCR assay for the specific detection of group B streptococci (GBS) and B. atrophaeus. The performance of the cellular IC was compared to that of a standard IC plasmid added to PCRs. Approximately 500 B. atrophaeus spores per PCR was found to be optimal since this did not interfere significantly with GBS detection for either DNA extraction method and yielded reproducible amplification and/or detection of B. atrophaeus genomic DNA serving as an IC template. Performance of the cellular IC was comparable to that of the standard IC. This novel IC system using nonpathogenic and hard-to-lyse B. atrophaeus spores allowed validation of both the DNA extraction procedure and the amplification and detection process. Use of a spore-based control also provides a universal control for microbial cell lysis.

  7. Performance of self-collected penile-meatal swabs compared to clinician-collected urethral swabs for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium by nucleic acid amplification assays.

    PubMed

    Dize, Laura; Barnes, Perry; Barnes, Mathilda; Hsieh, Yu-Hsiang; Marsiglia, Vincent; Duncan, Della; Hardick, Justin; Gaydos, Charlotte A

    2016-10-01

    Men were enrolled in a study to assess the performance and acceptability of self-collected penile meatal swabs as compared to clinician-collected urethral swabs for sexually transmitted infections (STIs). We expected penile-meatal swabs to perform favorably to urethral swabs for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) detection by nucleic acid amplification assays (NAATs). Of 203 swab pairs tested; for CT, penile-meatal swab sensitivity was 96.8% and specificity was 98.8%. NG sensitivity and specificity were 100% and 98.9%, respectively. For TV, sensitivity was 85.0% and specificity was 96.7%. For MG sensitivity and specificity were 79.3% and 99.4%, respectively. No significant statistical differences between sample type accuracy (CT: P=0.625; NG: P=0.248; TV: P=0.344; and MG: P=0.070) existed. Most men, 90.1%, reported self-collection of penile-meatal swabs as "Very Easy" or "Easy". Self-collected penile-meatal swabs appeared acceptable for NAAT STI detection and an acceptable collection method by men.

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

    PubMed Central

    Pinto, Márcia; Entringer, Aline Piovezan; Steffen, Ricardo; Trajman, Anete

    2015-01-01

    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. PMID:26785963

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

    ERIC Educational Resources Information Center

    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)

  10. Recombinase Polymerase Amplification Compared to Real-Time Polymerase Chain Reaction Test for the Detection of Fasciola hepatica in Human Stool.

    PubMed

    Cabada, Miguel M; Malaga, Jose L; Castellanos-Gonzalez, Alejandro; Bagwell, Kelli A; Naeger, Patrick A; Rogers, Hayley K; Maharsi, Safa; Mbaka, Maryann; White, A Clinton

    2017-02-08

    Fasciola hepatica is the most widely distributed trematode infection in the world. Control efforts may be hindered by the lack of diagnostic capacity especially in remote endemic areas. Polymerase chain reaction (PCR)-based methods offer high sensitivity and specificity but require expensive technology. However, the recombinase polymerase amplification (RPA) is an efficient isothermal method that eliminates the need for a thermal cycler and has a high deployment potential to resource-limited settings. We report on the characterization of RPA and PCR tests to detect Fasciola infection in clinical stool samples with low egg burdens. The sensitivity of the RPA and PCR were 87% and 66%, respectively. Both tests were 100% specific showing no cross-reactivity with trematode, cestode, or nematode parasites. In addition, RPA and PCR were able to detect 47% and 26% of infections not detected by microscopy, respectively. The RPA adapted to a lateral flow platform was more sensitive than gel-based detection of the reaction products. In conclusion, the Fasciola RPA is a highly sensitive and specific test to diagnose chronic infection using stool samples. The Fasciola RPA lateral flow has the potential for deployment to endemic areas after further characterization.

  11. Improved testing for CMT1A and HNPP using multiplex ligation-dependent probe amplification (MLPA) with rapid DNA preparations: comparison with the interphase FISH method.

    PubMed

    Slater, Howard; Bruno, Damien; Ren, Hua; La, Phung; Burgess, Trent; Hills, Louise; Nouri, Sara; Schouten, Jan; Choo, K H Andy

    2004-08-01

    Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are the two most common peripheral neuropathies, with incidences of about 1 in 2,500. Several techniques can be used to detect the typical 1.5-Mb duplication or deletion associated with these respective conditions, but none combines simplicity with high sensitivity. MLPA is a new technique for measuring sequence dosage. We have assessed its performance for the detection of the specific 1.5-Mb duplication/deletion by prospectively testing 50 patients referred with differential diagnoses of CMT or HNPP. Probes were designed to evaluate the TEKT3, PMP22, and COX10 genes within the CMT1A/HNPP region. We have compared the results with our existing fluorescence in situ hybridization (FISH) assay, which was performed in parallel. There was concordance of results for 49 patients. Of note, one patient showed an intermediate multiplex ligation-dependent probe amplification (MLPA) result with an abnormal FISH result, which is consistent with mosaicism. The assay works equally well with either purified DNA or rapid DNA preparations made by direct cell lysis. The use of the latter significantly reduces the cost of the assay. MLPA is a sensitive, specific, robust, and cost-effective technique suitable for fast, high-throughput testing and offers distinct advantages over other testing methods.

  12. Innovative role of statistics in acid rain performance testing

    SciTech Connect

    Warren-Hicks, W.; Etchison, T.; Lieberman, E.R.

    1995-12-31

    Title IV of the Clean Air Act Amendments (CAAAs) of 1990 mandated that affected electric utilities reduce sulfur dioxide (SO{sub 2}) and nitrogen oxide (NO{sub x}) emissions, the primary precursors of acidic deposition, and included an innovative market-based SO{sub 2} regulatory program. A central element of the Acid Rain Program is the requirement that affected utility units install CEMS. This paper describes how the Acid Rain Regulations incorporated statistical procedures in the performance tests for continuous emissions monitoring systems (CEMS) and how statistical analysis was used to assess the appropriateness, stringency, and potential impact of various performance tests and standards that were considered for inclusion in the Acid Rain Regulations. Described here is the statistical analysis that was used to set a relative accuracy standard, establish the calculation procedures for filling in missing data when a monitor malfunctions, and evaluate the performance tests applied to petitions for alternative monitoring systems. The paper concludes that the statistical evaluations of proposed provisions of the Acid Rain Regulations resulted in the adoption of performance tests and standards that were scientifically substantiated, workable, and effective.

  13. Concordance study between one-step nucleic acid amplification and morphologic techniques to detect lymph node metastasis in papillary carcinoma of the thyroid.

    PubMed

    del Carmen, Sofía; Gatius, Sonia; Franch-Arcas, Guzmán; Baena, José Antonio; Gonzalez, Oscar; Zafon, Carlos; Cuevas, Dolors; Valls, Joan; Pérez, Angustias; Martinez, Mercedes; Ros, Susana; Macías, Carmen García; Iglesias, Carmela; Matías-Guiu, Xavier; de Álava, Enrique

    2016-02-01

    Tumor resection in papillary thyroid carcinoma (PTC) is often accompanied by lymph node (LN) removal of the central and lateral cervical compartments. One-step nucleic acid amplification (OSNA) is a polymerase chain reaction-based technique that quantifies cytokeratin 19 (CK19) messenger RNA copies. Our aim is to assess the value of OSNA in detection of LN metastases in PTC, in comparison with imprints and microscopic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue. A total of 387 LNs from 37 patients were studied. From each half LN, 2 imprints were taken and analyzed with hematoxylin and eosin (H&E) and CK19 immunostaining. One half of the LN was submitted to OSNA and one half to FFPE processing and H&E and CK19 staining. For concordance analysis, every single LN was considered as a case. A group of 11 cases with discordant results between OSNA and H&E/CK19 FFPE sections were subjected to additional FFPE serial sectioning and H&E and CK19 staining. We found a high degree of concordance between the assays used, with sensitivities ranging from 0.81 to 0.95, and specificities ranging from 0.87 and 0.98. OSNA allowed upstaging of patients from pN0 to pN1, in comparison with standard pathologic analysis. Identification of a metastatic LN with more than 15000 CK19 messenger RNA copies predicted the presence of a second LN with macrometastasis (<5000 copies). In summary, the study shows that OSNA application in sentinel or suspicious LN may be helpful in assessing nodal status in PTC patients.

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

    PubMed

    Liu, Yi; Duan, Chunhong; Zhang, Chunxiu; Yang, Xiaomeng; Zhao, Yan; Dong, Rui; Zhou, Jiajing; Gai, Zhongtao

    2015-01-01

    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.

  15. Real-time nucleic acid sequence-based amplification (NASBA) assay targeting MIC1 for detection of Cryptosporidium parvum and Cryptosporidium hominis oocysts.

    PubMed

    Hønsvall, Birgitte K; Robertson, Lucy J

    2017-01-01

    Both Cryptosporidium parvum and Cryptosporidium hominis are often associated with cryptosporidiosis in humans, but whereas humans are the main host for C. hominis, C. parvum is zoonotic and able to infect a variety of species. The oocyst transmission stages of both species of parasites are morphologically identical and molecular techniques, usually polymerase chain reaction (PCR), are required to distinguish between oocysts detected by standard methods in environmental samples, such as water. In this study, we developed two primer sets for real-time nucleic acid sequence-based amplification (NASBA), targeting the MIC1 transcript in C. parvum (CpMIC1) and C. hominis (ChMIC1). Using these primer sets, we were not only able to detect low numbers of C. parvum and C. hominis oocysts (down to 5 oocysts in 10 μl, and down to 1 oocyst using diluted RNA samples), but also distinguish between them. One of the primer sets targeted an exon only occurring in CpMIC1, thereby providing a tool for distinguishing C. parvum from other Cryptosporidium species. Although mRNA has been suggested as a tool for assessing viability of Cryptosporidium oocysts, as it is short-lived and may have high transcription, this NASBA assay detected MIC1 mRNA in inactivated oocysts. RNA within the oocysts seems to be protected from degradation, even when the oocysts have been killed by heating or freeze-thawing. Thus, our approach detects both viable and non-viable oocysts, and RNA does not seem to be a suitable marker for assessing oocyst viability.

  16. Adipic gets the acid test as flue gas scrubber additive

    SciTech Connect

    Miller, I.R.

    1980-02-11

    The first full-scale demonstration of adipic acid for such use, to be conducted early in the summer of 1980 in a 200 MW power plant burning high-sulfur coal, is designed to clarify the costs and show how to reduce losses of adipic acid via degradation. Adipic acid improves SO/sub 2/ removal by acting as a buffer to limit the pH drop normally occurring at the gas-liquid interface so that the higher SO/sub 2/ concentration in the surface film improves liquid-phase mass transfer; it also promotes higher limestone utilization. Prepared by the Tennessee Valley Authority, a preliminary economic analysis for a 500 MW plant burning 4% sulfur coal indicates that the addition of 1500 ppM of adipic acid (limestone at $7/ton and the acid at $840/ton) would raise SO/sub 2/ removal from 90 to 95%, reduce the total capital investment from $41.5 to $39.5 million, and have a first year revenue requirement of $19.9 million vs. $20.9 million without the acid. The large-scale trial will also help clarify concern over unpleasant odors that have been reported at test sites of the limestone/adipic system; valeric acid has been identified as the cause.

  17. 21 CFR 862.1305 - Formiminoglutamic acid (FIGLU) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Formiminoglutamic acid (FIGLU) test system. 862.1305 Section 862.1305 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  18. 21 CFR 862.1305 - Formiminoglutamic acid (FIGLU) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Formiminoglutamic acid (FIGLU) test system. 862.1305 Section 862.1305 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  19. The role of anxiety sensitivity cognitive concerns in suicidal ideation: A test of the Depression-Distress Amplification Model in clinical outpatients.

    PubMed

    Norr, Aaron M; Allan, Nicholas P; Macatee, Richard J; Capron, Daniel W; Schmidt, Norman B

    2016-04-30

    Suicide constitutes a significant public health burden as global suicide rates continue to increase. Thus, it is crucial to identify malleable suicide risk factors to develop prevention protocols. Anxiety sensitivity, or a fear of anxiety-related sensations, is a potential malleable risk factor for the development of suicidal ideation. The Depression-Distress Amplification Model (DDAM) posits that the anxiety sensitivity cognitive concerns (ASCC) subfactor interacts with depressive symptoms to amplify the effects of depression and lead to suicidal ideation. The current study tested the DDAM across the two most widely-replicated factors of depressive symptoms (cognitive and affective/somatic) in comparison to a risk factor mediation model where ASCC are related to suicidal ideation via depressive symptoms. Participants included 295 clinical outpatients from a community clinic. The interaction between ASCC and depressive symptoms in the prediction of suicidal ideation was not significant for either cognitive or affective/somatic symptoms of depression. However, results revealed a significant indirect effect of ASCC through cognitive symptoms of depression in the prediction of suicidal ideation. These cross sectional findings are not consistent with the DDAM. Rather, the relationship may be better conceptualized with a model in which ASCC is related to suicidal ideation via cognitive symptoms of depression.

  20. Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

    PubMed

    Del Río, Jonathan Sabaté; Lobato, Ivan Magriñà; Mayboroda, Olena; Katakis, Ioanis; O'Sullivan, Ciara K

    2017-03-02

    Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10(-15) M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

  1. Clinical significance of expression of human cytomegalovirus pp67 late transcript in heart, lung, and bone marrow transplant recipients as determined by nucleic acid sequence-based amplification.

    PubMed

    Gerna, G; Baldanti, F; Middeldorp, J M; Furione, M; Zavattoni, M; Lilleri, D; Revello, M G

    1999-04-01

    Human cytomegalovirus (HCMV) infection was monitored retrospectively by qualitative determination of pp67 mRNA (a late viral transcript) by nucleic acid sequence-based amplification (NASBA) in a series of 50 transplant recipients, including 26 solid-organ (11 heart and 15 lung) transplant recipients (SOTRs) and 24 bone marrow transplant recipients (BMTRs). NASBA results were compared with those obtained by prospective quantitation of HCMV viremia and antigenemia and retrospective quantitation of DNA in leukocytes (leukoDNAemia). On the whole, 29 patients were NASBA positive, whereas 10 were NASBA negative, and the blood of 11 patients remained HCMV negative. NASBA detected HCMV infection before quantitation of viremia did but after quantitation of leukoDNAemia and antigenemia did. In NASBA-positive blood samples, median levels of viremia, antigenemia, and leukoDNAemia were significantly higher than the relevant levels detected in NASBA-negative HCMV-positive blood samples. By using the quantitation of leukoDNAemia as the "gold standard," the analytical sensitivity (47.3%), as well as the negative predictive value (68. 3%), of NASBA for the diagnosis of HCMV infection intermediate between that of antigenemia quantitation (analytical sensitivity, 72. 3%) and that of viremia quantitation (analytical sensitivity, 28.7%), while the specificity and the positive predictive value were high (90 to 100%). However, with respect to the clinically relevant antigenemia cutoff of >/=100 used in this study for the initiation of preemptive therapy in SOTRs with reactivated HCMV infection, the clinical sensitivity of NASBA reached 100%, with a specificity of 68. 9%. Upon the initiation of antigenemia quantitation-guided treatment, the actual median antigenemia level was 158 (range, 124 to 580) in SOTRs who had reactivated infection and who presented with NASBA positivity 3.5 +/- 2.6 days in advance and 13.5 (range, 1 to 270) in the group that included BMTRs and SOTRs who had primary

  2. 21 CFR 862.3580 - Lysergic acid diethylamide (LSD) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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,...

  3. 21 CFR 862.3580 - Lysergic acid diethylamide (LSD) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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,...

  4. 21 CFR 862.3580 - Lysergic acid diethylamide (LSD) test system.

    Code of Federal Regulations, 2010 CFR

    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,...

  5. 21 CFR 862.3580 - Lysergic acid diethylamide (LSD) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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,...

  6. 21 CFR 862.3580 - Lysergic acid diethylamide (LSD) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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,...

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

    SciTech Connect

    Carlson, C.D.; Delegard, C.H.; Burgeson, I.E.: Schmidt, A.J.; Bredt, P.R.; Silvers, K.L.

    1999-04-01

    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.

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

    PubMed Central

    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

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

    PubMed

    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.

  10. Human Cytomegalovirus Immediate-Early mRNA Detection by Nucleic Acid Sequence-Based Amplification as a New Parameter for Preemptive Therapy in Bone Marrow Transplant Recipients

    PubMed Central

    Gerna, Giuseppe; Baldanti, Fausto; Lilleri, Daniele; Parea, Maurizio; Alessandrino, Emilio; Pagani, Ambrogio; Locatelli, Franco; Middeldorp, Jaap; Revello, M. Grazia

    2000-01-01

    Human cytomegalovirus (HCMV) infection was monitored retrospectively by qualitative determination of immediate-early (IE) mRNA by nucleic acid sequence-based amplification (NASBA) in a series of 51 bone marrow transplant (BMT) recipients. The qualitative results for IE mRNA obtained by NASBA were compared with those obtained by prospective quantitation of HCMV viremia and antigenemia and retrospective quantitation of DNA in blood (DNAemia) by PCR as well as by qualitative determination of late pp67 mRNA by NASBA. On the whole, of the 39 HCMV-positive patients (all asymptomatic), HCMV was detected in 14 (35.9%) by quantitation of viremia, 15 (38.5%) by detection of pp67 mRNA by NASBA, 32 (82.1%) by quantitation of DNAemia, and 33 (84.6%) by quantitation of antigenemia, while HCMV was detected in 38 (97.4%) patients by detection of IE mRNA by NASBA. In the immunocompetent host, IE mRNA was not detected by NASBA in 100 blood donors or during reactivated infections in 30 breast-feeding mothers. Likewise, NASBA did not detect IE mRNA in 56 solid-organ transplant recipients in the first 21 days after transplantation. By using NASBA for detection of IE mRNA as the reference standard for detection of HCMV infection in blood samples, the diagnostic sensitivities were 67.7% for quantitation of DNAemia, 59.0% for quantitation of antigenemia, 18.3% for detection of pp67 mRNA by NASBA, and 16.0% for quantitation of viremia. Specificities and negative and positive predictive values were >90.0, >70.0, and >80.0%, respectively, for all four assays. The mean times to first HCMV detection after bone marrow transplantation were 37.7 ± 15.4 days for detection of IE mRNA by NASBA, 39.6 ± 15.6 days for quantitation of antigenemia, 40.9 ± 15.2 days for quantitation of DNAemia, and 43.7 ± 16.3 or 43.7 ± 17.5 days for quantitation of viremia and detection of pp67 mRNA by NASBA, respectively. On the whole, 31 BMT recipients received preemptive therapy by using confirmed antigenemia

  11. A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon.

    PubMed

    Wu, Dong; Xu, Huo; Shi, Haimei; Li, Weihong; Sun, Mengze; Wu, Zai-Sheng

    2017-03-08

    K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases.

  12. 21 CFR 862.1390 - 5-Hydroxyindole acetic acid/serotonin test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false 5-Hydroxyindole acetic acid/serotonin test system... Test Systems § 862.1390 5-Hydroxyindole acetic acid/serotonin test system. (a) Identification. A 5-hydroxyindole acetic acid/serotonin test system is a device intended to measure 5-hydroxyindole acetic...

  13. 21 CFR 862.1390 - 5-Hydroxyindole acetic acid/serotonin test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false 5-Hydroxyindole acetic acid/serotonin test system... Test Systems § 862.1390 5-Hydroxyindole acetic acid/serotonin test system. (a) Identification. A 5-hydroxyindole acetic acid/serotonin test system is a device intended to measure 5-hydroxyindole acetic...

  14. 21 CFR 862.1390 - 5-Hydroxyindole acetic acid/serotonin test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false 5-Hydroxyindole acetic acid/serotonin test system... Test Systems § 862.1390 5-Hydroxyindole acetic acid/serotonin test system. (a) Identification. A 5-hydroxyindole acetic acid/serotonin test system is a device intended to measure 5-hydroxyindole acetic...

  15. 21 CFR 862.1390 - 5-Hydroxyindole acetic acid/serotonin test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false 5-Hydroxyindole acetic acid/serotonin test system... Test Systems § 862.1390 5-Hydroxyindole acetic acid/serotonin test system. (a) Identification. A 5-hydroxyindole acetic acid/serotonin test system is a device intended to measure 5-hydroxyindole acetic...

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

    PubMed

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

    2016-05-15

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

  17. Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater.

    PubMed

    Stedtfeld, Robert D; Stedtfeld, Tiffany M; Samhan, Farag; Kanitkar, Yogendra H; Hatzinger, Paul B; Cupples, Alison M; Hashsham, Syed A

    2016-12-01

    Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater. Experiments tested amplification of DNA with and without crude lysis and varying concentrations of humic acid. Three separate field-able methods of biomass concentration with eight aquifer samples were also tested, comparing direct LAMP with traditional DNA extraction and quantitative PCR (qPCR). A new technique was developed where filters were amplified directly within disposable Gene-Z chips. The direct filter amplification (DFA) method eliminated an elution step and provided a detection limit of 10(2)Dehalobacter cells per 100mL. LAMP with crudely lysed Dehalobacter had a negligible effect on threshold time and sensitivity compared to lysed samples. The LAMP assay was more resilient than traditional qPCR to humic acid in sample, amplifying with up to 100mg per L of humic acid per reaction compared to 1mg per L for qPCR. Of the tested field-able concentrations methods, DFA had the lowest coefficient of variation among Dehalobacter spiked groundwater samples and lowest threshold time indicating high capture efficiency and low inhibition. While demonstrated with Dehalobacter, the DFA method can potentially be used for a number of applications requiring field-able, rapid (<60min) and highly sensitive quantification of microorganisms in environmental water samples.

  18. Molecular screening test in familial forms of cerebral cavernous malformation: the impact of the Multiplex Ligation-dependent Probe Amplification approach.

    PubMed

    Penco, Silvana; Ratti, Rachele; Bianchi, Elena; Citterio, Alberto; Patrosso, Maria Cristina; Marocchi, Alessandro; Tassi, Laura; La Camera, Alessandro; Collice, Massimo

    2009-05-01

    Object The purpose of this study was to underline the effectiveness of molecular analysis in cerebral cavernous angioma, with special attention to the familial forms. Methods Multiplex Ligation-dependent Probe Amplification analysis integrates the consecutive sequence analysis of the 3 genes (Krit1/CCM1, MGC4607/CCM2, and PDCD10/CCM3) known to be responsible for cerebral cavernous malformation lesions. Results The Multiplex Ligation-dependent Probe Amplification analysis revealed a new mutation, a heterozygous exon 9/10 deletion of Krit1, in the proband and in all affected family members. Conclusions The identification of the molecular defect allows physicians to screen family members at risk and to identify affected individuals before the onset of clinical symptoms caused by the presence of lesions.

  19. Enzyme-catalysed deposition of ultrathin silver shells on gold nanorods: a universal and highly efficient signal amplification strategy for translating immunoassay into a litmus-type test.

    PubMed

    Yang, Xinjian; Gao, Zhiqiang

    2015-04-25

    On the basis of enzyme-catalysed reduction of silver ions and consequent deposition of ultrathin silver shells on gold nanorods, a highly efficient signal amplification method for immunoassay is developed. For a model analyte prostate-specific antigen, a 10(4)-fold improvement over conventional enzyme-linked immunosorbent assay is accomplished by leveraging on the cumulative nature of the enzymatic reaction and the sensitive response of plasnomic gold nanorods to the deposition the silver shells.

  20. Principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons

    PubMed Central

    Weusten, Jos J. A. M.; Carpay, Wim M.; Oosterlaken, Tom A. M.; van Zuijlen, Martien C. A.; van de Wiel, Paul A.

    2002-01-01

    For quantitative NASBA-based viral load assays using homogeneous detection with molecular beacons, such as the NucliSens EasyQ HIV-1 assay, a quantitation algorithm is required. During the amplification process there is a constant growth in the concentration of amplicons to which the beacon can bind while generating a fluorescence signal. The overall fluorescence curve contains kinetic information on both amplicon formation and beacon binding, but only the former is relevant for quantitation. In the current paper, mathematical modeling of the relevant processes is used to develop an equation describing the fluorescence curve as a function of the amplification time and the relevant kinetic parameters. This equation allows reconstruction of RNA formation, which is characterized by an exponential increase in concentrations as long as the primer concentrations are not rate limiting and by linear growth over time after the primer pool is depleted. During the linear growth phase, the actual quantitation is based on assessing the amplicon formation rate from the viral RNA relative to that from a fixed amount of calibrator RNA. The quantitation procedure has been successfully applied in the NucliSens EasyQ HIV-1 assay. PMID:11884645

  1. 21 CFR 862.1187 - Conjugated sulfolithocholic acid (SLCG) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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....

  2. 21 CFR 862.1187 - Conjugated sulfolithocholic acid (SLCG) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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....

  3. 21 CFR 862.1187 - Conjugated sulfolithocholic acid (SLCG) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 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....

  4. 21 CFR 862.1187 - Conjugated sulfolithocholic acid (SLCG) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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....

  5. 21 CFR 862.1187 - Conjugated sulfolithocholic acid (SLCG) test system.

    Code of Federal Regulations, 2010 CFR

    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....

  6. Electricity-Free Amplification and Detection for Molecular Point-of-Care Diagnosis of HIV-1

    PubMed Central

    Singleton, Jered; Osborn, Jennifer L.; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens. PMID:25426953

  7. Electricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1.

    PubMed

    Singleton, Jered; Osborn, Jennifer L; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens.

  8. Early amplification options.

    PubMed

    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.

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

    PubMed

    Boyle, David S; McNerney, Ruth; Teng Low, Hwee; Leader, Brandon Troy; Pérez-Osorio, Ailyn C; Meyer, Jessica C; O'Sullivan, Denise M; Brooks, David G; Piepenburg, Olaf; Forrest, Matthew S

    2014-01-01

    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 (IS1081)were 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 assays

  10. Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase.

    PubMed

    Lamas-Maceiras, Mónica; Vaca, Inmaculada; Rodríguez, Esther; Casqueiro, Javier; Martín, Juan F

    2006-04-01

    A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phl gene was confirmed by reverse transcriptase-PCR. The phl gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phl protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phl in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35% increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phl gene resulted in a 40% decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phl gene restored normal levels of penicillin production and resistance to phenylacetic acid. The phenylacetyl-CoA ligase encoded by the phl gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phl protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phl protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal co-localization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.

  11. A modified PCR protocol for consistent amplification of fatty acid desaturase (FAD) alleles in marker-assisted backcross breeding for high oleic trait in peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High oleic acid, such as is found in olive oil, is desirable for the healthy cholesterol-lowering benefits. The oxidative stability of the oil with high oleic acid also gives longer “shelve life” for peanut products. These benefits drive the breeding effort toward developing high oleic peanuts worl...

  12. Chemical amplification based on fluid partitioning in an immiscible liquid

    DOEpatents

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

    2010-09-28

    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.

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

    PubMed

    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.

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

    SciTech Connect

    G. G. Loomis; A. P. Zdinak; M. A. Ewanic; J. J. Jessmore

    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 characteristic leach ing

  15. Questioning cochlear amplification

    NASA Astrophysics Data System (ADS)

    van der Heijden, Marcel; Versteegh, Corstiaen P. C.

    2015-12-01

    Thirty years ago it was hypothesized that motile processes inject mechanical energy into cochlear traveling waves. This mechanical amplification, alternatively described as negative damping, is invoked to explain both the sensitivity and the nonlinear compression of cochlear responses. There is a recent trend to present cochlear amplification as an established fact, even though the evidence is at most circumstantial and several thorny problems have remained unresolved. We analyze several of these issues, and present new basilar membrane recordings that allowed us to quantify cochlear energy flow. Specifically, we address the following questions: (1) Does auditory sensitivity require narrowband amplification? (2) Has the "RC problem" (lowpass filtering of outer hair cell receptor potential) been resolved? (3) Can OHC motility improve auditory sensitivity? (4) Is there a net power gain between neighboring locations on the basilar membrane? The analyses indicate that mechanical amplification in the cochlea is neither necessary nor useful, and that realizing it by known forms of motility would reduce sensitivity rather than enhance it. Finally, our experimental data show that the peaking of the traveling wave is realized by focusing the acoustic energy rather than amplifying it. (Abbreviations. BM: basilar membrane; CF: characteristic frequency; IHC: inner hair cell; ME: middle ear; MT; mechanotransducer; OHC: outer hair cell; SPL: sound pressure level.)

  16. Rapid molecular diagnostic test for Zika virus with low demands on sample preparation and instrumentation.

    PubMed

    Eboigbodin, Kevin E; Brummer, Mirko; Ojalehto, Tuomas; Hoser, Mark

    2016-12-01

    Zika virus has only recently gained attention due to recent large outbreaks worldwide. An easy to use nucleic acid amplification test could play an important role in the early detection of the infection and patient management. Here, we report a rapid and robust isothermal nucleic acid amplification assay for the detection of Zika virus. The method is cost-effective and compatible with portable instrumentation, enabling near patient testing and field use.

  17. Bilateral amplification and sound localization: then and now.

    PubMed

    Simon, Helen J

    2005-01-01

    This article is concerned with the evolution and pros and cons of bilateral amplification. Determining whether a bilateral hearing aid fitting is superior to that of a monaural hearing aid is a long-standing question; for this reason, the trend toward bilateral amplification has been slow. However, it is now assumed that bilateral amplification has significant advantages over monaural amplification in most cases, a view that is supported by our localization results. In this article, we will address the advantages of bilateral hearing aids and reveal some new localization data that show that most listeners with bilateral amplification, when tested unaided, as well as normal-hearing listeners manifested very high degrees of symmetry in their judgments of perceived angle while listeners who routinely use monaural amplification and those with asymmetric hearing loss had relatively large asymmetries. These data show that asymmetry in localization judgments is a much more sensitive indicator of abnormal localization ability than the magnitude of localization errors.

  18. Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

    PubMed

    Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

    2014-06-17

    Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems.

  19. Qualitative detection of avian influenza A (H5N1) viruses: a comparative evaluation of four real-time nucleic acid amplification methods.

    PubMed

    Chantratita, Wasun; Sukasem, Chonlaphat; Kaewpongsri, Supaporn; Srichunrusami, Chutatip; Pairoj, Wantanit; Thitithanyanont, Arunee; Chaichoune, Kridsada; Ratanakron, Parntep; Songserm, Thaweesak; Damrongwatanapokin, Sudarat; Landt, Olfert

    2008-01-01

    The aim of this study was to determine the performance of real-time amplification based methods - NASBA, TaqMan, RT-FRET, and RT-PCR LUXtrade mark formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003-2006, results showed that the NASBA (H5=98.2%, N1=96.3%), TaqMan (H5=98.2%, N1=96.3%) and FRET (H5=98.2%, N1=96.3%) had significantly higher rates of positive detection than LUX (H5=94.4%, N1=50.0%; P<0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at > or =100copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses.

  20. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

    PubMed Central

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-01-01

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi. PMID:27600711

  1. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant-Associated Fungi.

    PubMed

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-09-29

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.

  2. Species specific identification of spore-producing microbes using the gene sequence of small acid-soluble spore coat proteins for amplification based diagnostics

    DOEpatents

    McKinney, Nancy

    2002-01-01

    PCR (polymerase chain reaction) primers for the detection of certain Bacillus species, such as Bacillus anthracis. The primers specifically amplify only DNA found in the target species and can distinguish closely related species. Species-specific PCR primers for Bacillus anthracis, Bacillus globigii and Clostridium perfringens are disclosed. The primers are directed to unique sequences within sasp (small acid soluble protein) genes.

  3. Gravitomagnetic amplification in cosmology

    SciTech Connect

    Tsagas, Christos G.

    2010-02-15

    Magnetic fields interact with gravitational waves in various ways. We consider the coupling between the Weyl and the Maxwell fields in cosmology and study the effects of the former on the latter. The approach is fully analytical and the results are gauge invariant. We show that the nature and the outcome of the gravitomagnetic interaction depends on the electric properties of the cosmic medium. When the conductivity is high, gravitational waves reduce the standard (adiabatic) decay rate of the B field, leading to its superadiabatic amplification. In poorly conductive environments, on the other hand, Weyl-curvature distortions can result into the resonant amplification of large-scale cosmological magnetic fields. Driven by the gravitational waves, these B fields oscillate with an amplitude that is found to diverge when the wavelengths of the two sources coincide. We present technical and physical aspects of the gravitomagnetic interaction and discuss its potential implications.

  4. 21 CFR 862.1655 - Pyruvic acid test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... measure pyruvic acid (an intermediate compound in the metabolism of carbohydrate) in plasma. Measurements obtained by this device are used in the evaluation of electrolyte metabolism and in the diagnosis...

  5. Draft Test Guideline: Fish Acute Toxicity Mitigated By Humic Acid

    EPA Pesticide Factsheets

    The following draft test guideline is part of a series of test guidelines that have been developed by EPA for use in the testing of pesticides and toxic substances, and the development of test data for submission to the Agency for review.

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

    PubMed

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

    2016-01-01

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

  7. Standard test method for acidity of distillation residues or hydrocarbon liquids

    SciTech Connect

    Not Available

    1980-01-01

    This method covers the qualitative determination of the acidity of the distillation residue from a gasoline. The sample of distillation residue or hydrocarbon liquid is shaken with water and the aqueous layer tested for acidity to methyl orange. Some petroleum products are treated with mineral acid as part of the refining procedure. Obviously, any residual mineral acid in a petroleum product is undesirable. The absence of a positive indication in the test for acidity of the distillation residue or aqueous extract of a hydrocarbon liquid is an assurance of the care used in refining the fuel or solvent.

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

    PubMed

    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

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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

  11. Amplification of an MFS transporter encoding gene penT significantly stimulates penicillin production and enhances the sensitivity of Penicillium chrysogenum to phenylacetic acid.

    PubMed

    Yang, Jing; Xu, Xinxin; Liu, Gang

    2012-11-20

    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 compartmentation 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 12 transmembrane 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.

  12. Mass spectrometry signal amplification for ultrasensitive glycoprotein detection using gold nanoparticle as mass tag combined with boronic acid based isolation strategy.

    PubMed

    Liu, Minbo; Zhang, Lijuan; Xu, Yawei; Yang, Pengyuan; Lu, Haojie

    2013-07-25

    We describe a novel method for rapid and ultrasensitive detection of intact glycoproteins without enzymatic pretreatment which was commonly used in proteomic research. This method is based on using gold nanoparticle (AuNP) as signal tag in laser desorption/ionization mass spectrometry (LDI-MS) analysis combined with boronic acid assisted isolation strategy. Briefly speaking, target glycoproteins were firstly isolated from sample solution with boronic acid functionalized magnetic microparticles, and then the surface modified gold nanoparticles were added to covalently bind to the glycoproteins. After that, these AuNP tagged glycoproteins were eluted from magnetic microparticles and applied to LDI-MS analysis. The mass signal of AuNP rather than that of glycoprotein was detected and recorded in this strategy. Through data processing of different standard glycoproteins, we have demonstrated that the signal of AuNP could be used to quantitatively represent glycoprotein. This method allows femtomolar detection of intact glycoproteins. We believe that the successful validation of this method on three different kinds of glycoproteins suggests the potential use for tracking trace amount of target glycoproteins in real biological samples in the near future.

  13. Unilateral versus bilateral amplification for adults with impaired hearing.

    PubMed

    Walden, Therese C; Walden, Brian E

    2005-09-01

    This study compared unilateral and bilateral aided speech recognition in background noise in 28 patients being fitted with amplification. Aided QuickSIN (Quick Speech-in-Noise test) scores were obtained for bilateral amplification and for unilateral amplification in each ear. In addition, right-ear directed and left-ear directed recall on the Dichotic Digits Test (DDT) was obtained from each participant. Results revealed that the vast majority of patients obtained better speech recognition in background noise on the QuickSIN from unilateral amplification than from bilateral amplification. There was a greater tendency for bilateral amplification to have a deleterious effect among older patients. Most frequently, better aided QuickSIN performance was obtained in the right ear of participants, despite similar hearing thresholds in both ears. Finally, patients tended to perform better on the DDT in the ear that provided less SNR loss on the QuickSIN. Results suggest that bilateral amplification may not always be beneficial in every daily listening environment when background noise is present, and it may be advisable for patients wearing bilateral amplification to remove one hearing aid when difficulty is encountered understanding speech in background noise.

  14. Acid test: lipid antigens get into the groove.

    PubMed

    Kronenberg, Mitchell; Sullivan, Barbara A

    2008-06-01

    How do CD1 molecules load lipid antigens? In this issue of Immunity, Relloso et al. (2008) uncover how lysosomal pH targets amino acids in CD1b, causing it to open and attain a conformation more receptive to lipid antigens.

  15. 21 CFR 862.1509 - Methylmalonic acid (nonquantitative) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... 862.1509 Section 862.1509 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN.... The identification of methylmalonic acid in urine is used in the diagnosis and treatment of methylmalonic aciduria, a heritable metabolic disorder which, if untreated, may cause mental retardation....

  16. 21 CFR 862.1509 - Methylmalonic acid (nonquantitative) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... 862.1509 Section 862.1509 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN.... The identification of methylmalonic acid in urine is used in the diagnosis and treatment of methylmalonic aciduria, a heritable metabolic disorder which, if untreated, may cause mental retardation....

  17. 21 CFR 862.1509 - Methylmalonic acid (nonquantitative) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... 862.1509 Section 862.1509 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN.... The identification of methylmalonic acid in urine is used in the diagnosis and treatment of methylmalonic aciduria, a heritable metabolic disorder which, if untreated, may cause mental retardation....

  18. Nucleic acid testing for tuberculosis at the point-of-care in high-burden countries

    PubMed Central

    Niemz, Angelika; Boyle, David S

    2013-01-01

    Early diagnosis of tuberculosis (TB) facilitates appropriate treatment initiation and can limit the spread of this highly contagious disease. However, commonly used TB diagnostic methods are slow, often insensitive, cumbersome and inaccessible to most patients in TB endemic countries that lack necessary resources. This review discusses nucleic acid amplification technologies, which are being developed for rapid near patient TB diagnosis, that are in the market or undergoing clinical evaluation. They are based on PCR or isothermal methods and are implemented as manual assays or partially/fully integrated instrument systems, with associated tradeoffs between clinical performance, cost, robustness, quality assurance and usability in remote settings by minimally trained personnel. Unmet needs prevail for the identification of drug-resistant TB and for TB diagnosis in HIV-positive and pediatric patients. PMID:23153237

  19. Comparison of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma as Measured by the NucliSens Nucleic Acid Sequence-Based Amplification and Quantiplex Branched-DNA Assays

    PubMed Central

    Ginocchio, C. C.; Tetali, S.; Washburn, D.; Zhang, F.; Kaplan, M. H.

    1999-01-01

    This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although the Quantiplex and NucliSens values were significantly different (P < 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays: r = 0.912, P < 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively. PMID:10074556

  20. Comparison of levels of human immunodeficiency virus type 1 RNA in plasma as measured by the NucliSens nucleic acid sequence-based amplification and Quantiplex branched-DNA assays.

    PubMed

    Ginocchio, C C; Tetali, S; Washburn, D; Zhang, F; Kaplan, M H

    1999-04-01

    This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although the Quantiplex and NucliSens values were significantly different (P < 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays: r = 0.912, P < 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively.

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

    PubMed

    Kersting, Sebastian; Rausch, Valentina; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2014-01-01

    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. FigureThe combination of multiplex isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides.

  2. Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

    DTIC Science & Technology

    2013-01-01

    Zika virus; NT = not tested. †Successfully detected virus at the genus and species level. ‡Detected virus at the genus, but not at the species level...mosquito-only flavivirus; MTBEV = mammalian tick-borne encephalitis virus; POTV = Potiskum virus; RBV = Rio Bravo virus; TMUV = Tembusu virus; ZIKV = Zika

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

    PubMed Central

    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

  4. Coherent white light amplification

    DOEpatents

    Jovanovic, Igor; Barty, Christopher P.

    2004-05-25

    A system for coherent simultaneous amplification of a broad spectral range of light that includes an optical parametric amplifier and a source of a seed pulse is described. A first angular dispersive element is operatively connected to the source of a seed pulse. A first imaging telescope is operatively connected to the first angular dispersive element and operatively connected to the optical parametric amplifier. A source of a pump pulse is operatively connected to the optical parametric amplifier. A second imaging telescope is operatively connected to the optical parametric amplifier and a second angular dispersive element is operatively connected to the second imaging telescope.

  5. Integration of isothermal amplification methods in microfluidic devices: Recent advances.

    PubMed

    Giuffrida, Maria Chiara; Spoto, Giuseppe

    2017-04-15

    The integration of nucleic acids detection assays in microfluidic devices represents a highly promising approach for the development of convenient, cheap and efficient diagnostic tools for clinical, food safety and environmental monitoring applications. Such tools are expected to operate at the point-of-care and in resource-limited settings. The amplification of the target nucleic acid sequence represents a key step for the development of sensitive detection protocols. The integration in microfluidic devices of the most popular technology for nucleic acids amplifications, polymerase chain reaction (PCR), is significantly limited by the thermal cycling needed to obtain the target sequence amplification. This review provides an overview of recent advances in integration of isothermal amplification methods in microfluidic devices. Isothermal methods, that operate at constant temperature, have emerged as promising alternative to PCR and greatly simplify the implementation of amplification methods in point-of-care diagnostic devices and devices to be used in resource-limited settings. Possibilities offered by isothermal methods for digital droplet amplification are discussed.

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

    PubMed Central

    Hamzah, Hairul Hisham; Yusof, Nor Azah; Salleh, Abu Bakar; Bakar, Fatimah Abu

    2011-01-01

    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. PMID:22164018

  7. Camera-based ratiometric fluorescence transduction of nucleic acid hybridization with reagentless signal amplification on a paper-based platform using immobilized quantum dots as donors.

    PubMed

    Noor, M Omair; Krull, Ulrich J

    2014-10-21

    Paper-based diagnostic assays are gaining increasing popularity for their potential application in resource-limited settings and for point-of-care screening. Achievement of high sensitivity with precision and accuracy can be challenging when using paper substrates. Herein, we implement the red-green-blue color palette of a digital camera for quantitative ratiometric transduction of nucleic acid hybridization on a paper-based platform using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). A nonenzymatic and reagentless means of signal enhancement for QD-FRET assays on paper substrates is based on the use of dry paper substrates for data acquisition. This approach offered at least a 10-fold higher assay sensitivity and at least a 10-fold lower limit of detection (LOD) as compared to hydrated paper substrates. The surface of paper was modified with imidazole groups to assemble a transduction interface that consisted of immobilized QD-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as an acceptor. A hybridization event that brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs was responsible for a FRET-sensitized emission from the acceptor dye, which served as an analytical signal. A hand-held UV lamp was used as an excitation source and ratiometric analysis using an iPad camera was possible by a relative intensity analysis of the red (Cy3 photoluminescence (PL)) and green (gQD PL) color channels of the digital camera. For digital imaging using an iPad camera, the LOD of the assay in a sandwich format was 450 fmol with a dynamic range spanning 2 orders of magnitude, while an epifluorescence microscope detection platform offered a LOD of 30 fmol and a dynamic range spanning 3 orders of magnitude. The selectivity of the hybridization assay was demonstrated by detection of a single nucleotide polymorphism at a contrast ratio of 60:1. This work provides an

  8. Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

    PubMed Central

    Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

    2012-01-01

    Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a

  9. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

    PubMed Central

    2014-01-01

    Background Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. Methods A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. Results The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Conclusions Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in

  10. Capsule report: Adipic acid-enhanced lime/limestone test results at the EPA alkali scrubbing test facility

    SciTech Connect

    Burbank, D.A.; Wang, S.C.

    1982-04-01

    The fifth in a series of reports describing the results of the Shawnee Lime and Limestone Wet Scrubbing Test Program, the report describes the results of adipic acid-enhanced limestone wet scrubbing systems. A primary objective of the program was to enhance sulfur oxide removal and improve the reliability and economics of lime and limestone wet scrubbing systems by use of adipic acid as a chemical additive.

  11. Evidence of high-elevation amplification versus Arctic amplification

    PubMed Central

    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. PMID:26753547

  12. Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics.

    PubMed

    James, Ameh; Macdonald, Joanne

    2015-01-01

    Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase amplification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid amplification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.

  13. X-29 flight - Acid test for design predictions

    NASA Technical Reports Server (NTRS)

    Putnam, T. W.; Petersen, K. L.; Ishmael, S. D.; Sefic, W. J.

    1986-01-01

    The X-29 flight test data are being disseminated to interested industrial and military users as fast as it becomes available. The aircraft is extensively instrumented with accelerometers and pressure sensors and optical sensors for measuring wing deflection. The thoroughness of preflight preparations permitted a rapid advance through initial test checkpoints, which have both confirmed many predictions and revealed several discrepancies. The flight envelope had been expanded to Mach 1.1 and an altitude of 40,000 ft by December 1985. Notably, the X-29 has provided in-flight data which could not be faithfully depicted in a simulator, e.g., flare procedures during landing, and has shown that the stability adjustments, although adequate for controlling the aircraft, are not rapid enough to offer a satisfactory margin of harmony. The tests are now being performed in the transonic regime, where supercritical airfoil and forward swept wing drag reduction become significant factors.

  14. Novel DNA Polymer for Amplification Pretargeting

    PubMed Central

    2015-01-01

    In this Letter, different from conventional pretargeting, an additional novel DNA polymer with multiple copies of a target was first designed to be administrated between the antitumor antibody, and the labeled effector served as an amplification pretargeting strategy. Two phosphorothioate DNA strands, a bridging and a target strand, were hybridized to form a polymer. Polymer size, as a function of molar ratios, was then monitored by size exclusion HPLC and electrophoretic mobility shift assay. Moreover, binding efficiency of polymers with the radiolabeled effector and polymer size after hybridization were measured by HPLC as well. As the polymer was expected to produce more binding sites that would be targeted by effectors, amplification pretargeting can greatly improve accumulation of effectors in tumor. This novel proof-of-concept was then well demonstrated by the in vitro test of signal amplification in antibody-binding protein L coated plate and LS174T cells. Compared to conventional pretargeting, significantly increasing radioactive signal was observed in this designed amplification pretargeting, which would serve as a useful paradigm of the potential of oligomer polymers to improve pretargeting and other related approaches. PMID:26396682

  15. Hands-On Science: Is It an Acid or a Base? These Colorful Tests Tell All!

    ERIC Educational Resources Information Center

    VanCleave, Janice

    1998-01-01

    Two hands-on science activities for K-6 students teach them how to determine if something is an acid or a base. The activities require acid/base indicator juice, testing strips, and a base solution. A recipe for making them in the classroom using red cabbage and baking soda is provided. (SM)

  16. Detection of Human Cytomegalovirus pp67 Late Gene Transcripts in Cerebrospinal Fluid of Human Immunodeficiency Virus Type 1-Infected Patients by Nucleic Acid Sequence-Based Amplification

    PubMed Central

    Zhang, Fan; Tetali, Surya; Wang, Xue Ping; Kaplan, Mark H.; Cromme, Frans V.; Ginocchio, Christine C.

    2000-01-01

    This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82.7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease. PMID:10790122

  17. Detection of human cytomegalovirus pp67 late gene transcripts in cerebrospinal fluid of human immunodeficiency virus type 1-infected patients by nucleic acid sequence-based amplification.

    PubMed

    Zhang, F; Tetali, S; Wang, X P; Kaplan, M H; Cromme, F V; Ginocchio, C C

    2000-05-01

    This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82. 7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease.

  18. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  19. A new test procedure for biogenic sulfuric acid corrosion of concrete

    PubMed

    Vincke; Verstichel; Monteny; Verstraete

    1999-01-01

    A new test method is described for biogenic sulfuric acid corrosion of concrete, more specifically in sewer conditions. The aim of the new test method is the development of an accelerated and reproducible procedure for monitoring the resistance of different types of concrete with regard to biogenic sulfuric acid corrosion. This experimental procedure reflects worst case conditions by providing besides H2S, also an enrichment of thiobacilli and biologically produced sulfur. By simulating the cyclic processes occurring in sewer pipes, significant differences between concrete mixtures could be detected after 51 days. Concrete modified by a styrene-acrylic ester polymer demonstrated a higher resistance against biogenic sulfuric acid attack.

  20. Battery-triggered ultrasensitive electrochemiluminescence detection on microfluidic paper-based immunodevice based on dual-signal amplification strategy.

    PubMed

    Li, Weiping; Li, Meng; Ge, Shenguang; Yan, Mei; Huang, Jiadong; Yu, Jinghua

    2013-03-12

    Dual-signal amplification strategy for ultrasensitive electrochemiluminescence (ECL) multiplexed immunoassay on microfluidic paper-based analytical devices (μ-PADs) was demonstrated. This dual-signal amplification technique was achieved by employing graphene oxide-chitosan/gold nanoparticles (GCA) immunosensing platform and [4,4'-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid] (P-acid) functionalized nanoporous silver (P-acid/NPS) signal amplification label. For further low-cost and disposable applications, battery-triggered constant-potential ECL (+1.0V for P-acid label (vs. Ag/AgCl auxiliary electrode)) was applied on this paper-based immunodevice with the aid of a home-made voltage-tunable power device, allowing the traditional electrochemical workstation to be abandoned. We found that two tumor markers could be sequentially detected in the linear ranges of 0.003-20 and 0.001-10ngmL(-1) with the detection limits down to 1.0 and 0.8pgmL(-1), respectively, by simply reversing the connection mode on two working electrodes. The results exhibited excellent precision and high sensitivity of such immunoassay, and it also demonstrated that this battery-triggered ECL paper-based immunodevice could provide a rapid, simple and simultaneous multiplex immunoassay with high throughput, low-cost and low detection limits for point-of-care testing.

  1. 21 CFR 862.1055 - Newborn screening test system for amino acids, free carnitine, and acylcarnitines using tandem...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Newborn screening test system for amino acids... 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...

  2. 21 CFR 862.1055 - Newborn screening test system for amino acids, free carnitine, and acylcarnitines using tandem...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Newborn screening test system for amino acids... 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...

  3. 21 CFR 862.1055 - Newborn screening test system for amino acids, free carnitine, and acylcarnitines using tandem...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Newborn screening test system for amino acids... 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...

  4. 78 FR 58574 - Maintenance, Testing, and Replacement of Vented Lead-Acid Storage Batteries for Nuclear Power Plants

    Federal Register 2010, 2011, 2012, 2013, 2014

    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... with regard to the maintenance, testing, and replacement of vented lead-acid storage batteries...

  5. Testing the Role of Silicic Acid and Bioorganic Materials in the Formation of Rock Coatings

    SciTech Connect

    Kolb, Vera; Philip, Ajish I.; Perry, Randall S.

    2004-12-01

    Silica, amino acids, and DNA were recently discovered in desert varnish. In this work we experimentally test the proposed role of silicic acid and bio-chemicals in the formation of desert varnish and other rock coatings. We have developed a protocol in which hte rocks were treated with a mixture of silicic acid, sugars, amino acids, metals and clays, under the influence of heat and UV light. This protocol reflects the proposed mechanism of hte polymerization of silicic acid with the bioorganic materials, and the laboratory model for the natural conditions under which the desert varnish is formed. Our experiments produced coatings with a hardness and morphology that resemble the nature ones. These results provide a support for the role of silicic acid in the formation of rock coatings. Since the hard silica-based coatings preserve organic compounds in them, they may serve as a biosignature for life, here or possibly Mars.

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

    PubMed

    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.

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

    PubMed Central

    2016-01-01

    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

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

    SciTech Connect

    Carlson, C.D.; Delegard, C.H.; Burgeson, I.E.; Schmidt, A.J.; Silvers, K.L.

    1999-04-02

    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 {approximately}2.5 g of sludge (dry basis). The high solids loading test was conducted with {approximately}7 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.

  9. Equipment-Free Incubation of Recombinase Polymerase Amplification Reactions Using Body Heat

    PubMed Central

    Richards-Kortum, Rebecca

    2014-01-01

    The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings. PMID:25372030

  10. Equipment-free incubation of recombinase polymerase amplification reactions using body heat.

    PubMed

    Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

    2014-01-01

    The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings.

  11. Post-Fragmentation Whole Genome Amplification-Based Method

    NASA Technical Reports Server (NTRS)

    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 (<400 bp) constitute a library of 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

  12. A SIMPLE ASSAY FOR 2,4-DICHLOROPHENOXYACETIC ACID USING COATED TEST-STRIPS

    EPA Science Inventory

    Immunoassay test strips utilizing ascending chromatography has been devised for the detection of 2.4-dichlorophenoxyacetic acid (2,4-D). This test requires no instrumentation, inexpensive reagents and relies on the application of antibodies to 2,4-D adsorbed onto colloidal gol...

  13. Vibration test methods and their experimental research on the performance of the lead-acid battery

    NASA Astrophysics Data System (ADS)

    He, Baoxiang; Wang, Hua; He, Xie

    2014-12-01

    As we know, Lead-acid battery is difficult to balance many factors such as the accuracy and the on-line testing requirement. The detecting system, as stated in this article, is based on the vibration test procedure, dynamically following the electrochemical process of the Lead-acid Battery, and collects the real-time state parameters for calculation, analysis and judgment. It also quantizes precisely the degradation and chargeability of the battery and therefore self-adapts to the ideal target values. During the test, it has not charged and discharged large current to the lead-acid battery, it only plus a smaller and shorter time of impulse voltage signal on both ends of lead-acid battery, so the battery measured is damage free, and the system energy consumption is small; Using the load compensation technology, it has solved the influence of load on the test results. What's more, the load characteristics are improved at the same time, it realized the online detection. The vibration detection is based on the adaptive fuzzy inference model which has taken various factors into account, concerning the choices of input aspects which may influence the output value. It realized a number of Lead-acid Battery voltage self-adaption and accomplished a variety of high-precise tests.

  14. Cycle life testing of a 24-V, 15-Ah sealed lead-acid aircraft battery

    SciTech Connect

    Vutetakis, D.G.; Viswanathan, V.V.

    1997-12-01

    This paper presents the results of cycle life testing of 24-V, 15-Ah sealed lead-acid batteries intended for use in the B-1B aircraft. Test samples were procured from two different manufacturers and subjected to cycle testing at 33% and 100% depth-of-discharge (DOD). The cycle life at 33% DOD ranged from 500 to 750 cycles. The cycle life at 100% DOD ranged from 160 to 260 cycles.

  15. Centrosome Amplification Is Sufficient to Promote Spontaneous Tumorigenesis in Mammals.

    PubMed

    Levine, Michelle S; Bakker, Bjorn; Boeckx, Bram; Moyett, Julia; Lu, James; Vitre, Benjamin; Spierings, Diana C; Lansdorp, Peter M; Cleveland, Don W; Lambrechts, Diether; Foijer, Floris; Holland, Andrew J

    2017-02-06

    Centrosome amplification is a common feature of human tumors, but whether this is a cause or a consequence of cancer remains unclear. Here, we test the consequence of centrosome amplification by creating mice in which centrosome number can be chronically increased in the absence of additional genetic defects. We show that increasing centrosome number elevated tumor initiation in a mouse model of intestinal neoplasia. Most importantly, we demonstrate that supernumerary centrosomes are sufficient to drive aneuploidy and the development of spontaneous tumors in multiple tissues. Tumors arising from centrosome amplification exhibit frequent mitotic errors and possess complex karyotypes, recapitulating a common feature of human cancer. Together, our data support a direct causal relationship among centrosome amplification, genomic instability, and tumor development.

  16. A field test of the effect of acidic rain on ion balance in a woodland salamander

    SciTech Connect

    Frisbie, M.P.; Wyman, R.L. )

    1994-06-01

    Earlier laboratory studies demonstrated that red-backed salamanders, Plethodon cinereus, are susceptible to osmotic disruption by low pH substrates. In natural systems, however, acidic input from precipitation may be mediated by soils before it impacts salamanders. We tested the effect of acidic rain on sodium balance in salamanders by confining individuals in enclosure in two forest types (hemlock, beech) for 34 d. Enclosures received artificial rain of either pH 3 or 5 every 3-4 d. Soils inside enclosures in the hemlock forest were more acidic than those in the beech forest at the outset. At termination, [H[sup +

  17. Characterizing Corrosion Effects of Weak Organic Acids Using a Modified Bono Test

    NASA Astrophysics Data System (ADS)

    Zhou, Yuqin; Turbini, Laura J.; Ramjattan, Deepchand; Christian, Bev; Pritzker, Mark

    2013-12-01

    To meet environmental requirements and achieve benefits of cost-effective manufacturing, no-clean fluxes (NCFs) or low-solids fluxes have become popular in present electronic manufacturing processes. Weak organic acids (WOAs) as the activation ingredients in NCFs play an important role, especially in the current lead-free and halogen-free soldering technology era. However, no standard or uniform method exists to characterize the corrosion effects of WOAs on actual metallic circuits of printed wiring boards (PWBs). Hence, the development of an effective quantitative test method for evaluating the corrosion effects of WOAs on the PWB's metallic circuits is imperative. In this paper, the modified Bono test, which was developed to quantitatively examine the corrosion properties of flux residues, is used to characterize the corrosion effects of five WOAs (i.e., abietic acid, succinic acid, glutaric acid, adipic acid, and malic acid) on PWB metallic circuits. Experiments were performed under three temperature/humidity conditions (85°C/85% RH, 60°C/93% RH, and 40°C/93% RH) using two WOA solution concentrations. The different corrosion effects among the various WOAs were best reflected in the testing results at 40°C and 60°C. Optical microscopy was used to observe the morphology of the corroded copper tracks, and scanning electron microscopy (SEM) energy-dispersive x-ray (EDX) characterization was performed to determine the dendrite composition.

  18. Testing and commercialization of byproduct dibasic acids as buffer additives for limestone flue gas desulfurization systems

    SciTech Connect

    Chang, J.C.S.; Mobley, J.D.

    1983-10-01

    Pilot plant (0.1 MW) tests and utility boiler full scale demonstration (194 MW) of byproduct organic dibasic acids (DBA) as buffer additives to limestone scrubbers have shown performance improvements equivalent to those achieved by the addition of pure adipic acid. Both SO/sub 2/ removal efficiency and limestone utilization increased, and no significant operating problems were observed with three of the four DBA tested. Chemical and biological evaluations of scrubber samples taken during the DBA testing indicated no detectable toxicity or mutagenicity, and no significant environmental impact is expected as a result of DBA addition. Economic estimates indicate that substitution of DBA for pure adipic acid as a buffer additive will result in additive cost savings of 30% or greater.

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

    PubMed

    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.

  20. 78 FR 15753 - Maintenance, Testing, and Replacement of Vented Lead-Acid Storage Batteries for Nuclear Power Plants

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-12

    ... COMMISSION Maintenance, Testing, and Replacement of Vented Lead-Acid Storage Batteries for Nuclear Power..., DG-1269 ``Maintenance, Testing, and Replacement of Vented Lead-Acid Storage Batteries for Nuclear... lead-acid storage batteries in nuclear power plants. DATES: Submit comments by May 13, 2013....

  1. Self-primed isothermal amplification for genomic DNA detection of human papillomavirus.

    PubMed

    Lu, Wei; Yuan, Qingpan; Yang, Zhiliu; Yao, Bo

    2017-04-15

    Rolling circle amplification (RCA) is an isothermal amplification technique with high efficiency and perfect accuracy for nucleic acids detection. However, RCA technique suffers the limitation to detect short DNA or RNA molecules. For long nucleic acid molecules, enzymatic restriction as well as heat denaturation process is usually required, which makes the amplification not effective and strictly isothermal. In this article, a simple and efficient one-pot self-primed isothermal amplification (SIA) was developed for detection of genomic DNA directly based on the combination of nicking endonuclease assisted strand displacement amplification (SDA) and exponential RCA. In virtue of numerous nicking sites on the genome, a pre-amplification of the whole genome was performed through SDA with the specific cleaving of nicking endonuclease. Meanwhile, the single strand DNA with HPV target sequence generated from SDA could hybrid with the circle probe as a primer and trigger the exponential RCA as a result of the existence of nicking endonuclease. As the reaction temperature and enzyme were the same, the amplification could be operated in one pot. The reaction solution after amplification was added on the electrode for hybridization with the sulfydryl probe to achieve the electrochemical signal. Based on the isothermal amplification, genotyping of HPV 11, 16, 18 and the detection of HPV 18 in Hela cell line were attempted with satisfied results. This approach should be a promising tool for pathogene detection in clinical diagnostics and research.

  2. [Test for detection of activated partial thromboplastin time using ellagic acid].

    PubMed

    Berkovskiĭ, A L; Sergeeva, E V; Kachalova, N D; Prostakova, T M; Kozlov, A A

    1999-06-01

    A simple and sensitive method for estimation of activated partial thromboplastin time (APTT) is developed, making use a complex reagent containing the activator (plant phospholipids) and contact factor (ellagic acid). The test requires additionally only 0.025 M CaCl2. The test is more sensitive to the presence of heparin in the blood and to insufficiency of blood clotting factors VIII and IX than the reagents containing insoluble substances (kaolin and animal phosphatides). Addition of soluble ellagic acid into reagent for APTT estimation allows studies on optic coagulometers.

  3. Detection of the food allergen celery via loop-mediated isothermal amplification technique.

    PubMed

    Zahradnik, Celine; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

    2014-11-01

    Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0% for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.

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

    PubMed

    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.

  5. Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses.

    PubMed

    Moore, Matthew D; Jaykus, Lee-Ann

    2017-01-09

    Human norovirus is a leading cause of viral gastroenteritis worldwide. Rapid detection could facilitate control, however widespread point-of-care testing is infrequently done due to the lack of robust and portable methods. Recombinase polymerase amplification (RPA) is a novel isothermal method which rapidly amplifies and detects nucleic acids using a simple device in near real-time. An RT-RPA assay targeting a recent epidemic human norovirus strain (GII.4 New Orleans) was developed and evaluated in this study. The assay successfully detected purified norovirus RNA from multiple patient outbreak isolates and had a limit of detection of 3.40 ± 0.20 log10 genomic copies (LGC), which is comparable to most other reported isothermal norovirus amplification methods. The assay also detected norovirus in directly boiled stool, and displayed better resistance to inhibitors than a commonly used RT-qPCR assay. The assay was specific, as it did not amplify genomes from 9 non-related enteric viruses and bacteria. The assay detected norovirus in some samples in as little as 6 min, and the entire detection process can be performed in less than 30 min. The reported RT-RPA method shows promise for sensitive point-of-care detection of epidemic human norovirus, and is the fastest human norovirus amplification method to date.

  6. Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

    PubMed Central

    Moore, Matthew D.; Jaykus, Lee-Ann

    2017-01-01

    Human norovirus is a leading cause of viral gastroenteritis worldwide. Rapid detection could facilitate control, however widespread point-of-care testing is infrequently done due to the lack of robust and portable methods. Recombinase polymerase amplification (RPA) is a novel isothermal method which rapidly amplifies and detects nucleic acids using a simple device in near real-time. An RT-RPA assay targeting a recent epidemic human norovirus strain (GII.4 New Orleans) was developed and evaluated in this study. The assay successfully detected purified norovirus RNA from multiple patient outbreak isolates and had a limit of detection of 3.40 ± 0.20 log10 genomic copies (LGC), which is comparable to most other reported isothermal norovirus amplification methods. The assay also detected norovirus in directly boiled stool, and displayed better resistance to inhibitors than a commonly used RT-qPCR assay. The assay was specific, as it did not amplify genomes from 9 non-related enteric viruses and bacteria. The assay detected norovirus in some samples in as little as 6 min, and the entire detection process can be performed in less than 30 min. The reported RT-RPA method shows promise for sensitive point-of-care detection of epidemic human norovirus, and is the fastest human norovirus amplification method to date. PMID:28067278

  7. Experiments on the abiotic amplification of optical activity

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Blair, N. E.; Dirbas, F. M.

    1981-01-01

    Experiments concerning the physical mechanisms for the abiotic generation and chemical mechanisms for the amplification of optical activity in biological compounds are reviewed. Attention is given to experiments involving the determination of the differential adsorption of racemic amino acids on d- and l-quartz, the asymmetric photolysis of racemic amino acids by circularly polarized light, and the asymmetric radiolysis of solid amino acids by longitudinally polarized electrons, and the enantiomeric enrichments thus obtained are noted. Further experiments on the amplification of the chirality in the polymerization of D, L-amino acid mixtures and the hydrolysis of D-, L-, and D, L-polypeptides are discussed. It is suggested that a repetitive cycle of partial polymerization-hydrolyses may account for the abiotic genesis of optically enriched polypeptides on the primitive earth.

  8. [Recombinase Polymerase Amplification and its Applications in Parasite Detection].

    PubMed

    ZHENG, Wen-bin; WU, Yao-dong; MA, Jian-gang; ZHU, Xing-quan; ZHOU, Dong-hui

    2015-10-01

    Recombinase polymerase amplification (RPA) is a recently -developed isothermal nucleic-acid-amplification technology that is based on the nucleic acid replication mechanism in T4 bacteriophage. With this technique, nucleic-acid templates can be amplified to measurable levels within 20 min at 37-42 °C. The. RPA process has high sensitivity and specificity, and is simple to operate, thus nucleic acids can be detected rapidly in non-laboratory conditions. Since its development in 2006, the RPA technique has been applied in agriculture, food safety, medicine, transgene detection, etc. In this review, we will give an overview on the research progress of RPA and its application in parasite detection.

  9. Isolated etioplasts as test system for inhibitors of fatty acid biosynthesis

    SciTech Connect

    Lichtenthaler, H.K.; Kobek, K. )

    1989-04-01

    Isolated intact chloroplasts of mono- and dicotyledonous plants possess the capacity for de novo fatty acid biosynthesis, starting from {sup 14}C-acetate. These can be taken as test system for herbicides affecting fatty acid biosynthesis as shown earlier in our laboratory. The incorporation rates of acetate into the total fatty acids depend on the photosynthetic cofactors ATP and NADPH and amount in the light to 33 kBq (oat) and 39 kBq (pea) per mg chlorophyll x h, whereas in the dark only ca. 10% of these rates are obtained. In order to establish a test system, which is fully independent of light, we isolated and characterized etioplast fractions from oat and pea seedlings with a very high capacity of de novo fatty acid biosynthesis (500 and 400 kBq per mg carotenoids in a 20 min period). This activity was blocked by herbicides such as cycloxydim, sethoxydim and diclofop in a dose-dependent manner. This new test system has the great advantage that one can verify whether inhibitors of photosynthesis affect fatty acid biosynthesis.

  10. Use of ramification amplification assay for detection of Escherichia coli O157:H7 and other E. coli Shiga toxin-producing strains.

    PubMed

    Li, Fan; Zhao, Chunyan; Zhang, Wandi; Cui, Shenghui; Meng, Jianghong; Wu, Josephine; Zhang, David Y

    2005-12-01

    Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx(2)). Our results showed that as few as 10 copies of stx(2) could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx(2) gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.

  11. Detection of biological molecules using chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2001-01-01

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal. Specifically, the analyte transducer immobilized in a polymeric matrix can be a boronic acid moiety.

  12. Chromosomal destabilization during gene amplification.

    PubMed Central

    Ruiz, J C; Wahl, G M

    1990-01-01

    Acentric extrachromosomal elements, such as submicroscopic autonomously replicating circular molecules (episomes) and double minute chromosomes, are common early, and in some cases initial, intermediates of gene amplification in many drug-resistant and tumor cell lines. In order to gain a more complete understanding of the amplification process, we investigated the molecular mechanisms by which such extrachromosomal elements are generated and we traced the fate of these amplification intermediates over time. The model system consists of a Chinese hamster cell line (L46) created by gene transfer in which the initial amplification product was shown previously to be an unstable extrachromosomal element containing an inverted duplication spanning more than 160 kilobases (J. C. Ruiz and G. M. Wahl, Mol. Cell. Biol. 8:4302-4313, 1988). In this study, we show that these molecules were formed by a process involving chromosomal deletion. Fluorescence in situ hybridization was performed at multiple time points on cells with amplified sequences. These studies reveal that the extrachromosomal molecules rapidly integrate into chromosomes, often near or at telomeres, and once integrated, the amplified sequences are themselves unstable. These data provide a molecular and cytogenetic chronology for gene amplification in this model system; an early event involves deletion to generate extrachromosomal elements, and subsequent integration of these elements precipitates a cascade of chromosome instability. Images PMID:2188107

  13. Acid rain program: CEMS submission instructions for monitoring plans, certification test notifications, and quarterly reports

    SciTech Connect

    1995-05-12

    The Acid Rain Program regulations require all affected utility units to continuously measure, record and report SO2, NOx, volumetric flow data and CO2 emissions. All affected units also must continuously measure and record opacity, and must report opacity exceedances to the appropriate State or Local Agency. To ensure that your CEMS and fuel flowmeters are performing at an acceptable level, and providing quality assured data, you are required under 40 CFR 75.53, 75.62 (a) to submit a monitoring plan and certification test data for acid rain CEM certificaton. The purpose of this handbook is to help you fulfill your requirements under the Acid Rain Program. This handbook will walk you through the necessary steps for gaining CEMS certification, including filling out and mailing the proper forms, administering the required tests, and applying for certification and sending in electronic data to EPA.

  14. Column leaching test to evaluate the use of alkaline industrial wastes to neutralize acid mine tailings

    SciTech Connect

    Doye, I.; Duchesne, J.

    2005-08-01

    Acid mine drainage is a serious environmental problem caused by the oxidation of sulfide minerals that releases highly acidic, sulfate, and metals-rich drainage. In this study, alkaline industrial wastes were mixed with acid mine tailings in order to obtain neutral conditions. A series of column leaching tests were performed to evaluate the behavior of reactive mine tailings amended with alkaline-additions under dynamic conditions. Column tests were conducted of oxidized mine tailings combined with cement kiln dust, red mud bauxite, and mixtures of cement kiln dust with red mud bauxite. The pH results show the addition of 10% of alkaline materials permits the maintenance of near neutral conditions. In the presence of 10% alkaline material, the concentration of toxic metals such as Al, Cu, Fe, Zn are significantly reduced as well as the number of viable cells (Thiobacillus ferrooxidans) compared to control samples.

  15. Misidentification of Mycobacterium leprae as Mycobacterium intracellulare by the COBAS AMPLICOR M. intracellulare Test

    PubMed Central

    Lefmann, M.; Moter, A.; Schweickert, B.; Göbel, U. B.

    2005-01-01

    Commercially available nucleic acid probe- and amplification-based systems for detection and differentiation of mycobacteria are widely used in clinical microbiology laboratories. Here we report two cases of human leprosy in which the COBAS AMPLICOR Mycobacterium intracellulare test led to false- positive results. Correct identification of Mycobacterium leprae was possible only by amplification and comparative sequence analysis of the 16S rRNA gene. PMID:15815021

  16. Direct amplification of casework bloodstains using the Promega PowerPlex(®) 21 PCR amplification system.

    PubMed

    Gray, Kerryn; Crowle, Damian; Scott, Pam

    2014-09-01

    A significant number of evidence items submitted to Forensic Science Service Tasmania (FSST) are blood swabs or bloodstained items. Samples from these items routinely undergo phenol:chloroform:isoamyl alcohol organic extraction and quantitative Polymerase Chain Reaction (qPCR) testing prior to PowerPlex(®) 21 amplification. This multi-step process has significant cost and timeframe implications in a fiscal climate of tightening government budgets, pressure towards improved operating efficiencies, and an increasing emphasis on rapid techniques better supporting intelligence-led policing. Direct amplification of blood and buccal cells on cloth and Whatman FTA™ card with PowerPlex(®) 21 has already been successfully implemented for reference samples, eliminating the requirement for sample pre-treatment. Scope for expanding this method to include less pristine casework blood swabs and samples from bloodstained items was explored in an endeavour to eliminate lengthy DNA extraction, purification and qPCR steps for a wider subset of samples. Blood was deposited onto a range of substrates including those historically found to inhibit STR amplification. Samples were collected with micro-punch, micro-swab, or both. The potential for further fiscal savings via reduced volume amplifications was assessed by amplifying all samples at full and reduced volume (25 and 13μL). Overall success rate data showed 80% of samples yielded a complete profile at reduced volume, compared to 78% at full volume. Particularly high success rates were observed for the blood on fabric/textile category with 100% of micro-punch samples yielding complete profiles at reduced volume and 85% at full volume. Following the success of this trial, direct amplification of suitable casework blood samples has been implemented at reduced volume. Significant benefits have been experienced, most noticeably where results from crucial items have been provided to police investigators prior to interview of

  17. Decomposition of hydroxy amino acids in foraminiferal tests; kinetics, mechanism and geochronological implications

    USGS Publications Warehouse

    Bada, J.L.; Shou, M.-Y.; Man, E.H.; Schroeder, R.A.

    1978-01-01

    The diagenesis of the hydroxy amino acids serine and threonine in foraminiferal tests has been investigated. The decomposition pathways of these amino acids are complex; the principal reactions appear to be dehydration, aldol cleavage and decarboxylation. Stereochemical studies indicate that the ??-amino-n-butyric acid (ABA) detected in foraminiferal tests is the end product of threonine dehydration pathway. Decomposition of serine and threonine in foraminiferal tests from two well-dated Caribbean deep-sea cores, P6304-8 and -9, has been found to follow irreversible first-order kinetics. Three empirical equations were derived for the disappearance of serine and threonine and the appearance of ABA. These equations can be used as a new geochronological method for dating foraminiferal tests from other deep-sea sediments. Preliminary results suggest that ages deduced from the ABA kinetics equation are most reliable because "species effect" and contamination problems are not important for this nonbiological amino acid. Because of the variable serine and threonine contents of modern foraminiferal species, it is likely that the accurate age estimates can be obtained from the serine and threonine decomposition equations only if a homogeneous species assemblage or single species sample isolated from mixed natural assemblages is used. ?? 1978.

  18. Classroom Demonstration of a Spot Test for Pbenylpyruvic Acid and Its Relationship to Phenylketonuria

    ERIC Educational Resources Information Center

    Halkides, Christopher J.

    2004-01-01

    Classical phenylketonuria (PKU) is caused by a lack activity in the enzyme phenylalanine hydroxylase, leading to elevated concentrations of phenylalanine in the blood. A simple demonstration and three advanced demonstrations of a spot test for phenylpyruvic acid and its relationship to phenylketonuria are given.

  19. 21 CFR 862.1060 - Delta-aminolevulinic acid test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

  20. 21 CFR 862.1060 - Delta-aminolevulinic acid test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

  1. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  2. 21 CFR 862.1390 - 5-Hydroxyindole acetic acid/serotonin test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false 5-Hydroxyindole acetic acid/serotonin test system. 862.1390 Section 862.1390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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...

  4. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  5. 21 CFR 862.1255 - 2,3-Diphosphoglyceric acid test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

  6. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  7. 21 CFR 862.1060 - Delta-aminolevulinic acid test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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...

  8. 21 CFR 862.1060 - Delta-aminolevulinic acid test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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...

  9. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  10. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  11. 21 CFR 862.1255 - 2,3-Diphosphoglyceric acid test system.

    Code of Federal Regulations, 2010 CFR

    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...

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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.

    Code of Federal Regulations, 2010 CFR

    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. Testing for departures from additivity in mixtures of perfluoroalkyl acids (PFAAs)

    EPA Science Inventory

    This study is a follow-up to a paper by Carr, et al. that determined a design structure to optimally test for departures from additivity in a fixed ratio mixture of four perfluoroalkyl acids (PFAAs) using an in vitro transiently-transfected COS- 1 PPARa reporter model with an NHA...

  15. 21 CFR 862.1255 - 2,3-Diphosphoglyceric acid test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

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

    PubMed Central

    Abd El Wahed, Ahmed; Patel, Pranav; Faye, Oumar; Thaloengsok, Sasikanya; Heidenreich, Doris; Matangkasombut, Ponpan; Manopwisedjaroen, Khajohnpong; Sakuntabhai, Anavaj; Sall, Amadou A.; Hufert, Frank T.; Weidmann, Manfred

    2015-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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. PMID:26075598

  17. 75 FR 22814 - Guidance for Industry: Nucleic Acid Testing (NAT) for Human Immunodeficiency Virus Type 1 (HIV-1...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-30

    ...: 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 Deferral... Immunodeficiency Virus Type 1 (HIV-1) Nucleic Acid Test (NAT) and Hepatitis C Virus (HCV) NAT, on...

  18. Single-use, electricity-free amplification device for detection of HIV-1.

    PubMed

    Curtis, Kelly A; Rudolph, Donna L; Morrison, Daphne; Guelig, Dylan; Diesburg, Steven; McAdams, David; Burton, Robert A; LaBarre, Paul; Owen, Michele

    2016-11-01

    Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at -20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC.

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Sequence independent amplification of DNA

    DOEpatents

    Bohlander, Stefan K.

    1998-01-01

    The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei.

  1. Sequence independent amplification of DNA

    DOEpatents

    Bohlander, S.K.

    1998-03-24

    The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example, the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei. 25 figs.

  2. Apparatus for chemical amplification based on fluid partitioning in an immiscible liquid

    DOEpatents

    Anderson, Brian L [Lodi, CA; Colston, Bill W [San Ramon, CA; Elkin, Christopher J [San Ramon, CA

    2012-05-08

    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.

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

    DOEpatents

    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.

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

    DOEpatents

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

    2017-02-28

    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.

  5. A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid.

    PubMed

    Xue, Qingwang; Lv, Yanqin; Cui, Hui; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

    2015-01-26

    An autonomous DNA nanomachine based on rolling circle amplification (RCA)-bridged two-stage exonuclease III (Exo III)-induced recycling amplification (Exo III-RCA-Exo III) was developed for label-free and highly sensitive homogeneous multi-amplified detection of DNA combined with sensitive fluorescence detection technique. According to the configuration, the analysis of DNA is accomplished by recognizing the target to a unlabeled molecular beacon (UMB) that integrates target-binding and signal transducer within one multifunctional design, followed by the target-binding of UMB in duplex DNA removed stepwise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer that initiate RCA process with a rational designed padlock DNA. The RCA products containing thousands of repeated catalytic sequences catalytically hybridize with a hairpin reporter probe that includes a "caged" inactive G-quadruplex sequence (HGP) and were then detected by Exo III-assisted recycling amplification, liberating the active G-quadruplex and generating remarkable ZnPPIX/G-quadruplex fluorescence signals with the help of zinc(II)-protoporphyrin IX (ZnPPIX). The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.51 aM. In addition, this designed protocol can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases.

  6. Precipitation of PEG/Carboxyl-Modified Gold Nanoparticles with Magnesium Pyrophosphate: A New Platform for Real-Time Monitoring of Loop-Mediated Isothermal Amplification.

    PubMed

    Qin, Ailin; Fu, Lok Tin; Wong, Jacky K F; Chau, Li Yin; Yip, Shea Ping; Lee, Thomas M H

    2017-03-29

    Gold nanoparticles have proven to be promising for decentralized nucleic acid testing by virtue of their simple visual readout and absorbance-based quantification. A major challenge toward their practical application is to achieve ultrasensitive detection without compromising simplicity. The conventional strategy of thermocycling amplification is unfavorable (because of both instrumentation and preparation of thermostable oligonucleotide-modified gold nanoparticle probes). Herein, on the basis of a previously unreported co-precipitation phenomenon between thiolated poly(ethylene glycol)/11-mercaptoundecanoic acid co-modified gold nanoparticles and magnesium pyrophosphate crystals (an isothermal DNA amplification reaction byproduct), a new ultrasensitive and simple DNA assay platform is developed. The binding mechanism underlying the co-precipitation phenomenon is found to be caused by the complexation of carboxyl and pyrophosphate with free magnesium ions. Remarkably, poly(ethylene glycol) does not hinder the binding and effectively stabilizes gold nanoparticles against magnesium ion-induced aggregation (without pyrophosphate). In fact, a similar phenomenon is observed in other poly(ethylene glycol)- and carboxyl-containing nanomaterials. When the gold nanoparticle probe is incorporated into a loop-mediated isothermal amplification reaction, it remains as a red dispersion for a negative sample (in the absence of a target DNA sequence) but appears as a red precipitate for a positive sample (in the presence of a target). This results in a first-of-its-kind gold nanoparticle-based DNA assay platform with isothermal amplification and real-time monitoring capabilities.

  7. Microwave amplification with nanomechanical resonators.

    PubMed

    Massel, F; Heikkilä, T T; Pirkkalainen, J-M; Cho, S U; Saloniemi, H; Hakonen, P J; Sillanpää, M A

    2011-12-14

    The sensitive measurement of electrical signals is at the heart of modern technology. According to the principles of quantum mechanics, any detector or amplifier necessarily adds a certain amount of noise to the signal, equal to at least the noise added by quantum fluctuations. This quantum limit of added noise has nearly been reached in superconducting devices that take advantage of nonlinearities in Josephson junctions. Here we introduce the concept of the amplification of microwave signals using mechanical oscillation, which seems likely to enable quantum-limited operation. We drive a nanomechanical resonator with a radiation pressure force, and provide an experimental demonstration and an analytical description of how a signal input to a microwave cavity induces coherent stimulated emission and, consequently, signal amplification. This generic scheme, which is based on two linear oscillators, has the advantage of being conceptually and practically simpler than the Josephson junction devices. In our device, we achieve signal amplification of 25 decibels with the addition of 20 quanta of noise, which is consistent with the expected amount of added noise. The generality of the model allows for realization in other physical systems as well, and we anticipate that near-quantum-limited mechanical microwave amplification will soon be feasible in various applications involving integrated electrical circuits.

  8. Microsatellites Cross-Species Amplification across Some African Cichlids.

    PubMed

    Bezault, Etienne; Rognon, Xavier; Gharbi, Karim; Baroiller, Jean-Francois; Chevassus, Bernard

    2012-01-01

    The transfer of the genomic resources developed in the Nile tilapia, Oreochromis niloticus, to other Tilapiines sensu lato and African cichlid would provide new possibilities to study this amazing group from genetics, ecology, evolution, aquaculture, and conservation point of view. We tested the cross-species amplification of 32 O. niloticus microsatellite markers in a panel of 15 species from 5 different African cichlid tribes: Oreochromines (Oreochromis, Sarotherodon), Boreotilapiines (Tilapia), Chromidotilapines, Hemichromines, and Haplochromines. Amplification was successfully observed for 29 markers (91%), with a frequency of polymorphic (P(95)) loci per species around 70%. The mean number of alleles per locus and species was 3.2 but varied from 3.7 within Oreochromis species to 1.6 within the nontilapia species. The high level of cross-species amplification and polymorphism of the microsatellite markers tested in this study provides powerful tools for a wide range of molecular genetic studies within tilapia species as well as for other African cichlids.

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

    PubMed

    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.

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

    SciTech Connect

    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.

  11. The amplification of polymerized diaminobenzidine with physical developers: sensitizing effects of transition metal salts and sulphide.

    PubMed

    von Ruhland, C J; Jasani, B

    2010-05-01

    Amplification of metal-complexed polymerized diaminobenzidine by two light-insensitive physical developers was systematically examined in a dot blot model system following either polymerizing diaminobenzidine in the presence of transition metal salts or applying the metal salts post-diaminobenzidine polymerization. The effect of sodium sulphide treatment on subsequent amplification was also investigated. Those metal-diaminobenzidine complexes that facilitated the most powerful amplification were subsequently tested in an immunohistochemical setting. The most dramatic amplification of polymerized diaminobenzidine was observed following its post-polymerization treatment with salts of platinum alone, or gold or vanadium with subsequent sulphide treatment, and allowed previously invisible quantities of polymerized diaminobenzidine to be clearly seen. Three other transition metal salts also improved the amplification of polymerized diaminobenzidine but to a lesser degree, namely nickel alone, and silver or rhodium with subsequent sulphide treatment. Sensitivity was comparable with the colloidal gold-silver amplification system.

  12. Optical chirped beam amplification and propagation

    DOEpatents

    Barty, Christopher P.

    2004-10-12

    A short pulse laser system uses dispersive optics in a chirped-beam amplification architecture to produce high peak power pulses and high peak intensities without the potential for intensity dependent damage to downstream optical components after amplification.

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

    PubMed

    Hoser, Mark J; Mansukoski, Hannu K; Morrical, Scott W; Eboigbodin, Kevin E

    2014-01-01

    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.

  14. Sequence dependence of isothermal DNA amplification via EXPAR

    PubMed Central

    Qian, Jifeng; Ferguson, Tanya M.; Shinde, Deepali N.; Ramírez-Borrero, Alissa J.; Hintze, Arend; Adami, Christoph; Niemz, Angelika

    2012-01-01

    Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67–70% sensitivity and 77–80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions. PMID:22416064

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

    PubMed

    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.

  16. Controlled Microwave Heating Accelerates Rolling Circle Amplification.

    PubMed

    Yoshimura, Takeo; Suzuki, Takamasa; Mineki, Shigeru; Ohuchi, Shokichi

    2015-01-01

    Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.

  17. 21 CFR 862.1055 - Newborn screening test system for amino acids, free carnitine, and acylcarnitines using tandem...

    Code of Federal Regulations, 2010 CFR

    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...

  18. Classroom Amplification To Enhance Student Performance.

    ERIC Educational Resources Information Center

    DiSarno, Neil J.; Schowalter, Melissa; Grassa, Patricia

    2002-01-01

    Discussion of classroom amplification systems to improve the performance of students with hearing loss or learning disabilities addresses the auditory challenges of inclusive classrooms, changing the classroom environment to reduce noise, types of amplification systems, and what teachers observe about amplification. (Contains references.) (DB)

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

    SciTech Connect

    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

  20. Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses

    PubMed Central

    Pavšič, Jernej; Parkes, Helen; Schimmel, Heinz; Foy, Carole A.; Karczmarczyk, Maria; Gutiérrez-Aguirre, Ion; Honeyborne, Isobella; Huggett, Jim F.; McHugh, Timothy D.; Milavec, Mojca; Zeichhardt, Heinz; Žel, Jana

    2014-01-01

    Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability. PMID:25392365

  1. Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses.

    PubMed

    Pavšič, Jernej; Devonshire, Alison S; Parkes, Helen; Schimmel, Heinz; Foy, Carole A; Karczmarczyk, Maria; Gutiérrez-Aguirre, Ion; Honeyborne, Isobella; Huggett, Jim F; McHugh, Timothy D; Milavec, Mojca; Zeichhardt, Heinz; Žel, Jana

    2015-07-01

    Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability.

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Update on Malaria Diagnostics and Test Utilization.

    PubMed

    Mathison, Blaine A; Pritt, Bobbi S

    2017-04-12

    Malaria is a potentially life-threatening disease requiring rapid diagnosis and treatment. Although microscopic examination of thick and thin blood films remains the gold standard for laboratory diagnosis, rapid antigen tests and nucleic acid amplification methods may also play a useful role for detection of acute infection. This review discusses the advantages and disadvantages of the commonly-used diagnostic methods and provides important practice points for optimal malaria test utilization.

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

    SciTech Connect

    Pool, K.H.; Delegard, C.H.; Schmidt, A.J.; Thornton, B.M.; Silvers, K.L.

    1999-04-02

    Approximately 73 m{sup 3} 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.

  5. Sound Field Amplification and Listening Behaviour in the Classroom.

    ERIC Educational Resources Information Center

    McSporran, Eileen; Butterworth, Yvonne; Rowson, Vivienne J.

    1997-01-01

    Asserts that the acoustical environment of the classroom is an important variable in the listening and psychoeducational function, both for children with hearing loss and those with normal hearing. Reports the results of a test of a sound amplification system in two primary classes; indicates the benefits of the system. (DSK)

  6. Numerical computations of turbulence amplification in shock wave interactions

    NASA Technical Reports Server (NTRS)

    Zang, T. A.; Hussaini, M. Y.; Bushnell, D. M.

    1983-01-01

    Numerical computations are presented which illustrate and test various effects pertinent to the amplification and generation of turbulence in shock wave turbulent boundary layer interactions. Several fundamental physical mechanisms are identified. Idealizations of these processes are examined by nonlinear numerical calculations. The results enable some limits to be placed on the range of validity of existing linear theories.

  7. QuantiLyse: reliable DNA amplification from single cells.

    PubMed

    Pierce, Kenneth E; Rice, John E; Sanchez, J Aquiles; Wangh, Lawrence J

    2002-05-01

    Amplification of DNA sequencesfrom single cells via PCR is increasingly used in basic research and clinical diagnostics but remains technically difficult. We have developed a cell lysis protocol that uses an optimized proteinase K solution, named QuantiLyse and permits reliable amplification from individual cells. This protocol was compared to other published methods by means of real-time PCR with molecular beacons. The results demonstrate that QuantiLyse treatment of single lymphocytes renders gene targets more availablefor amplification than other published proteinase K methods or lysis in water. QuantiLyse and an optimized alkaline lysis were equally effective in terms of target availability, although QuantiLyse offers greaterflexibility, as it does not require neutralization and can comprise a higher percentage of the final PCR volume. Maximum gene target availability is also obtained following QuantiLyse treatment of samples containing up to 10000 cells (the largest number tested). Thus, QuantiLyse maximizes the chances that targeted DNA sequences will be available for amplification during the first cycle of PCR, thereby reducing the variability among replicate reactions as well as the likelihood of amplification failure or allele drop-out. QuantiLyse will be useful in a range of investigations aimed at gene detection in small numbers of cells.

  8. Frequency domain optical parametric amplification

    PubMed Central

    Schmidt, Bruno E.; Thiré, Nicolas; Boivin, Maxime; Laramée, Antoine; Poitras, François; Lebrun, Guy; Ozaki, Tsuneyuki; Ibrahim, Heide; Légaré, François

    2014-01-01

    Today’s ultrafast lasers operate at the physical limits of optical materials to reach extreme performances. Amplification of single-cycle laser pulses with their corresponding octave-spanning spectra still remains a formidable challenge since the universal dilemma of gain narrowing sets limits for both real level pumped amplifiers as well as parametric amplifiers. We demonstrate that employing parametric amplification in the frequency domain rather than in time domain opens up new design opportunities for ultrafast laser science, with the potential to generate single-cycle multi-terawatt pulses. Fundamental restrictions arising from phase mismatch and damage threshold of nonlinear laser crystals are not only circumvented but also exploited to produce a synergy between increased seed spectrum and increased pump energy. This concept was successfully demonstrated by generating carrier envelope phase stable, 1.43 mJ two-cycle pulses at 1.8 μm wavelength. PMID:24805968

  9. Hybrid chirped pulse amplification system

    SciTech Connect

    Barty, Christopher P.; Jovanovic, Igor

    2005-03-29

    A hybrid chirped pulse amplification system wherein a short-pulse oscillator generates an oscillator pulse. The oscillator pulse is stretched to produce a stretched oscillator seed pulse. A pump laser generates a pump laser pulse. The stretched oscillator seed pulse and the pump laser pulse are directed into an optical parametric amplifier producing an optical parametric amplifier output amplified signal pulse and an optical parametric amplifier output unconverted pump pulse. The optical parametric amplifier output amplified signal pulse and the optical parametric amplifier output laser pulse are directed into a laser amplifier producing a laser amplifier output pulse. The laser amplifier output pulse is compressed to produce a recompressed hybrid chirped pulse amplification pulse.

  10. Performance assessment of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex from respiratory and extrapulmonary specimens.

    PubMed

    Piersimoni, Claudio; Scarparo, Claudio; Piccoli, Paola; Rigon, Alessandra; Ruggiero, Giuliana; Nista, Domenico; Bornigia, Stefano

    2002-11-01

    The new BDProbeTec ET Mycobacterium tuberculosis Complex Direct Detection Assay (DTB) was compared with the enhanced M. tuberculosis Amplified Direct Test (AMTDII). The system is an automated walkaway system characterized by simultaneous DNA amplification (strand displacement amplification) and real-time fluorometric detection. It also contains an internal amplification control (IAC) designed to identify inhibition from the processed samples. The AMTDII assay amplifies rRNA by transcription-mediated amplification; it uses hybridization with a chemoluminescent probe as a detection system and is entirely manual. A total of 515 N-acetyl-L-cysteine-sodium hydroxide-decontaminated respiratory (n = 331) and extrapulmonary (n = 184) sediments (from 402 patients) were tested in parallel by both assays. The results were compared with those of acid-fast staining and culture (solid plus liquid media), setting the combination of culture and clinical diagnosis as the "gold standard." Culture results from the tested specimens were as follows: 121 Mycobacterium tuberculosis complex (MTB) (98 smear-positive), 46 nontuberculous mycobacteria (38 smear-positive), and 338 culture-negative results. After resolution of the discrepant results, the percent sensitivity, percent specificity, and positive and negative likelihood ratios for AMTDII were 88%, 99.2%, 110, and 0.11 for respiratory specimens and 74.3%, 100%, 740, and 0.26 for extrapulmonary specimens, respectively. The corresponding values for DTB were 94.5%, 99.6%, 235, and 0.05 for respiratory specimens and 92.3%, 100%, 920, and 0.07 for extrapulmonary specimens, respectively. The cumulative difference for all tuberculosis-positive extrapulmonary specimens was significant (P = 0.03). The overall inhibition rate for DTB was 5% (26 specimens). We conclude that both amplification assays proved to be rapid and specific for the detection of MTB in clinical samples and particularly feasible for a routine laboratory work flow. DTB

  11. Rapid amplification of the RM-Yplex assay.

    PubMed

    Abuidrees, Aqeela S; Alghafri, Rashed H; Hadi, Sibte

    2016-10-01

    A multiplex PCR assay consisting of 13 Rapidly Mutating Y STR loci called RM-Yplex was previously developed. Platinum(®) Taq DNA polymerase was used to amplify the 13 Y STR loci in a single reaction at an amplification time of approximately 2.5 h. In order to shorten the process with reliable results, two DNA polymerases were tested with the multiplex. Phusion(®) Flash High Fidelity, TAKARA Z-taq(TM) , and Platinum(®) Taq DNA polymerases were investigated for conducting RM-Yplex assay at various PCR cycling conditions. Rapid, robust, and efficient amplification of all the markers within the multiplex were achieved. The amplification time was reduced from 2.5 h to less than 28 min with Phusion(®) Flash High Fidelity DNA polymerase using Veriti(®) PCR thermal cycler.

  12. Ecotoxicity of boric acid in standard laboratory tests with plants and soil organisms.

    PubMed

    Princz, Juliska; Becker, Leonie; Scheffczyk, Adam; Stephenson, Gladys; Scroggins, Rick; Moser, Thomas; Römbke, Jörg

    2017-03-17

    To verify the continuous sensitivity of ecotoxicological tests (mainly the test organisms), reference substances with known toxicity are regularly tested. Ideally, this substance(s) would lack specificity in its mode action, be bioavailable and readily attainable with cost-effective means of chemical characterization. Boric acid has satisfied these criteria, but has most recently been characterized as a substance of very high concern, due to reproductive effects in humans, thus limiting its recommendation as an ideal reference toxicant. However, there is probably no other chemical for which ecotoxicity in soil has been so intensively studied; an extensive literature review yielded lethal (including avoidance) and sublethal data for 38 taxa. The ecotoxicity data were evaluated using species sensitivity distributions, collectively across all taxa, and separately according to species type, endpoints, soil type and duration. The lack of specificity in the mode of action yielded broad toxicity among soil taxa and soil types, and provided a collective approach to assessing species sensitivity, while taking into consideration differences in test methodologies and exposure durations. Toxicity was species-specific with Folsomia candida and enchytraied species demonstrating the most sensitivity; among plants, the following trend occurred: dicotyledonous (more sensitive) ≫ monocotyledonous ≫ gymnosperm species. Sensitivity was also time and endpoint specific, with endpoints such as lethality and avoidance being less sensitive than reproduction effects. Furthermore, given the breadth of data and toxicity demonstrated by boric acid, lessons learned from its evaluation are discussed to recommend the properties required by an ideal reference substance for the soil compartment.

  13. Nucleic acid detection and quantification in the developing world.

    PubMed

    Huggett, Jim; Green, Clare; Zumla, Alimuddin

    2009-04-01

    Techniques using nucleic acid amplification have not had the same amount of impact on research and clinical diagnosis in the developing world as that observed in the West. This is unsurprising when the costs and infrastructure required to perform nucleic acid amplification are considered. Despite this, nucleic acid amplification is being increasingly used in both research and diagnosis in countries such as Zambia and Tanzania. Scientific research in the developing world is made possible through the support and development of the necessary laboratory infrastructure and the establishment of special transport for the reagents and samples. This has enabled world-leading country-relevant research to be performed by local scientists on subjects ranging from rapid diagnosis of infectious diseases to measuring the RNA gene expression in an immune response. Concomitantly, the challenge presented by the need for tests that are more appropriate for a resource-poor setting has led to a number of newer methodologies for nucleic acid detection, which can be tailored to be performed in the field without the need for training in molecular biology. As nucleic acid amplification techniques become both simpler and cheaper, their impact is likely to play an increasingly crucial role in research and diagnosis in the developing world.

  14. Deterministic noiseless amplification of coherent states

    NASA Astrophysics Data System (ADS)

    Hu, Meng-Jun; Zhang, Yong-Sheng

    2015-08-01

    A universal deterministic noiseless quantum amplifier has been shown to be impossible. However, probabilistic noiseless amplification of a certain set of states is physically permissible. Regarding quantum state amplification as quantum state transformation, we show that deterministic noiseless amplification of coherent states chosen from a proper set is attainable. The relation between input coherent states and gain of amplification for deterministic noiseless amplification is thus derived. Furthermore, we extend our result to more general situation and show that deterministic noiseless amplification of Gaussian states is also possible. As an example of application, we find that our amplification model can obtain better performance in homodyne detection to measure the phase of state selected from a certain set. Besides, other possible applications are also discussed.

  15. Development and validation of dissolution testings in acidic media for rabeprazole sodium delayed-release capsules

    PubMed Central

    Tan, Yinhe; Si, Xiaoqing; Zhong, Lulu; Feng, Xin; Yang, Xinmin; Huang, Min; Wu, Chuanbin

    2016-01-01

    Abstract Rabeprazole sodium (RAB) dissolved in acidic media is accompanied by its degradation in the course of dissolution testing. To develop and establish the accumulative release profiles of ACIPHEX® Sprinkle (RAB) delayed-release capsules (ACIPHEX® Sprinkle) in acidic media using USP apparatus 2 (paddle apparatus) as a dissolution tester, the issues of determination of accumulative release amount of RAB in these acidic media and interference of hydroxypropylmethyl cellulose phthalate were solved by adding appropriate hydrochloric acid (HCl) into dissolution samples coupled with centrifugation so as to remove the interference and form a solution of degradation products of RAB, which is of a considerably stable ultraviolet (UV) absorbance at the wavelength of 298 nm within 2.0 h. Therefore, the accumulative release amount of RAB in dissolution samples at each sample time points could be determined by UV-spectrophotometry, and the accumulative release profiles of ACIPHEX® Sprinkle in the media of pH 1.0, pH 6.0, and pH 6.8 could be established. The method was validated per as the ICH Q2 (R1) guidelines and demonstrated to be adequate for quality control of ACIPHEX® Sprinkle and the accumulative release profiles can be used as a tool to guide the formulation development and quality control of a generic drug for ACIPHEX® Sprinkle. PMID:27066697

  16. Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests

    PubMed Central

    Karlberg, Helen; Bragstad, Karoline; Lindegren, Gunnel; Stoltz, Malin Lundahl; Salata, Cristiano; Kran, Anne-Marte Bakken; Dudman, Susanne Gjeruldsen; Mirazimi, Ali; Fomsgaard, Anders

    2016-01-01

    Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport, and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available Magna Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding Magna Pure lysis/binding buffer directly into vacuum blood collection EDTA tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months, and Ebola virus RNA is preserved in the Magna Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from Magna Pure lysis/binding buffer-inactivated samples using the QIAamp viral RNA minikit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe, and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection. PMID:27466385

  17. Rolling circle amplification detection of RNA and DNA

    DOEpatents

    Christian, Allen T.; Pattee, Melissa S.; Attix, Cristina M.; Tucker, James D.

    2004-08-31

    Rolling circle amplification (RCA) has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate RCA in solution and in situ to detect gene copy number and single base mutations. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.

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

    PubMed Central

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

    2016-01-01

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

  19. Evaluation of a semiquantitative SNAP test for measurement of bile acids in dogs.

    PubMed

    Seibert, Rachel L; Tobias, Karen M; Reed, Ann; Snyder, Karl R

    2014-01-01

    Background. Serum bile acids (SBA) are used as a routine screening tool of liver function in dogs. Serum samples are usually shipped to a referral laboratory for quantitative analysis with an enzymatic chemistry analyzer. The canine SNAP Bile Acids Test (SNAP-BAT) provides an immediate, semi-quantitative measurement of bile acid concentrations in-house. With the SNAP-BAT, bile acids concentrations of 5-30 µmol/L are quantified, and results outside of that range are classified as <5 or >30 µmol/L. Agreement of the SNAP-BAT with the enzymatic method has not been extensively investigated. Objectives. The purposes of this prospective clinical study were to assess the precision of the SNAP-BAT and determine agreement of SNAP-BAT with results from an in-house chemistry analyzer. Methods. After verifying intra-assay precision of the SNAP-BAT, a prospective analysis was performed using blood samples collected from 56 dogs suspected to have liver disease. Each sample was analyzed with an enzymatic, in-house chemistry analyzer and the SNAP-BAT. Agreement between the two methods was statistically assessed using the κ index of agreement. Results. Intra-assay variability was minimal. The κ index for agreement between the SNAP-BAT and routine chemistry analyzer was between 0.752 and 0.819, indicating substantial to near perfect agreement. Conclusions. The SNAP-BAT is a highly accurate, semi-quantitative test that yields immediate results, and has very little intra-assay variability, particularly for results >30 µmol/L.

  20. Chelidonic acid evokes antidepressant-like effect through the up-regulation of BDNF in forced swimming test

    PubMed Central

    Jeong, Hyun-Ja; Yang, Shi-Young; Kim, Hee-Yun; Kim, Na-Rae; Jang, Jae-Bum

    2016-01-01

    Depression is usually accompanied by neuro-inflammatory reactions. Chelidonic acid, in particular, has shown anti-inflammatory effects. The objective of this study was to evaluate the anti-depressant effects of chelidonic acid and to discuss the potential mechanisms of a forced swimming test. Chelidonic acid was administered orally once a day for 14 days. On the 14th day, chelidonic acid resulted in a significant decrease in immobility time during the forced swimming test without alteration of locomotor activity, in an open field test. Chelidonic acid also increased the number of nissl bodies in the hippocampus. Brain-derived neurotrophic factor expression and extracellular signal-regulated protein kinase phosphorylation in the hippocampus were up-regulated by the administration of chelidonic acid. Chelidonic acid administration significantly increased the mRNA expression of hippocampal estrogen receptor-β. The levels of hippocampal interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were effectively attenuated by the administration of chelidonic acid. In addition, chelidonic acid significantly increased the levels of 5-hydroxytryptamine (serotonin), dopamine, and norepinephrine compared with those levels for the mice that were administered distilled water in the hippocampus. These results suggest that chelidonic acid might serve as a new therapeutic strategy for the regulation of depression associated with inflammation. PMID:27037280

  1. Calibration of fusidic acid disk diffusion susceptibility testing of Staphylococcus areus.

    PubMed

    Olsson-Liljequist, Barbro; Kõljalg, Siiri; Karlsson, Inga; Kronvall, Göran

    2002-10-01

    Single strain regression analysis, SRA, was used to calibrate disk diffusion fusidic acid susceptibility testing of Staphylococcus aureus in two laboratories using different standard methods but the same interpretative MIC limits. SRA equation constants were calculated using five different fusidic acid disk contents (1.5, 5, 15, 50, 150 microg). These disks were tested on five separate occasions against quality control strain S. aureus ATCC 29213. The National Committee for Clinical Laboratory Standards (NCCLS) method was employed in Tartu, Estonia (TE) and the Swedish Reference Group for Antibiotics (SRGA) method in Sweden at the Karolinska Hospital (KS). SRA constants obtained were used for calculating zone breakpoints corresponding to MIC breakpoints recommended by the SRGA (S < or = 0.5 mg/L, R > or = 1 mg/L). Zone diameter histograms from KS, performed with a 50 microg disk, and from TE, using a 10 microg disk, showed a clustering of wild type strains around 41 mm and 30 mm, respectively, reflecting differences in methodology. Zone breakpoints calculated from the equations were validated by comparison with the histograms. Breakpoints were also calculated for a suggested lower disk content in Sweden, 10 microg, and validated in tests of clinical isolates and by histogram analysis.

  2. Toxicological assessment of refined naphthenic acids in a repeated dose/developmental toxicity screening test.

    PubMed

    McKee, Richard H; North, Colin M; Podhasky, Paula; Charlap, Jeffrey H; Kuhl, Adam

    2014-01-01

    Naphthenic acids (NAs) are primarily cycloaliphatic carboxylic acids with 10 to 16 carbons. To characterize the potential of refined NAs (>70% purity) to cause reproductive and/or developmental effects, Sprague-Dawley rats (12/group) were given oral doses of 100, 300, or 900 mg/kg/d, beginning 14 days prior to mating, then an additional 14 days for males or through lactation day 3 for females (up to 53 days) in a repeated dose/reproductive toxicity test (Organization for Economic Cooperation and Development [OECD] 422). Potential mutagenic effects were assessed using Salmonella (OECD 471) and in in vivo micronucleus tests (OECD 474) using bone marrow taken from treated animals in the screening study described previously. Systemic effects included reduced terminal body weights, increased liver weights, and changes in a number of blood cell parameters. The overall no effect level for all target organ effects was 100 mg/kg/d. In the reproductive/developmental toxicity assessment, there were significant reductions in numbers of live born offspring in groups exposed to 300 and 900 mg/kg/d. The overall no effect level for developmental effects was 100 mg/kg/d. The data from the Salmonella and micronucleus tests provide evidence that refined NAs are not genotoxic.

  3. [Nondestructive test on predicting sugar content and valid acidity of mango by spectroscopy technology].

    PubMed

    Yu, Jia-jia; He, Yong; Bao, Yi-dan

    2008-12-01

    Mango is a kind of popular tropic fruit in the word, and its quality will affect the health of consumers. Unsaturated acid is an important component in mango. So it is very important and necessary to detect the sugar content and valid acidity in mango fast and non-destructively. Visible and short-wave near-infrared reflectance spectroscopy (VIS/SWNIRS) was applied in the present study to predict sugar content and valid acidity of mango. Because of the non-linear information in spectral data characteristics of the pattern were analyzed by neural network optimized by genetic algorithm (GA-BP). Spectral data were compressed by the partial least squares (PLS). The best number of principal components (PCs) was selected according the accumulative reliabilities (AR). PCs could be used to replace the complex spectral data. After some preprocessing and through full cross validation, 17 principal components presenting important information of spectra were confirmed as the best number of principal components for valid acidity, and 18 PCs as best number of principal components for sugar content. Then, these best principal components were taken as the input of GA-BP neural network. One hundred thirty five samples were randomly collected as modeling, and the remaining 45 as samples to check the forecast results by the model. For the sake of testing the GA-BP model, at the same time we took the BP neural network on the same PCs. The quality of the calibration model was evaluated by the correlation coefficients (R) and standard error of calibration (SECV), and the prediction results were assessed by correlation coefficients (R) and standard error of prediction (SEP). Comparing PLS-BP model with PLS-GA-BP model, the coefficients of determination (R) of 0.788/0.83699 and standard errors of prediction (SEP) of 0.133312/0.109447 were calculated in valid acidity. The sugar content result was calculated by the coefficients of determination (R) = 0.75705/0.85409 and standard errors of

  4. Specific primer amplification of the VP1 region directed by 5' UTR sequence analysis: enterovirus testing and identification in clinical samples from hand-foot-and-mouth disease patients.

    PubMed

    Ge, Shengxiang; Yan, Qiang; He, Shuizhen; Zhuang, Sijie; Niu, Jianjun; Xia, Ningshao

    2013-11-01

    Many genotypes of the enterovirus (EV) pathogens can cause clinical hand-foot-and-mouth disease (HFMD). Therefore, rapid identification and monitoring of HFMD pathogens can be difficult, especially from the original clinical specimens. In this study, both universal pan-enterovirus and EV71/CA16 VP1-specific primer sets were designed and used to examine clinical specimens from HFMD patients. Based on the initial sequence analysis of the 5'-untanslated region (5'-UTR) and VP1 amplification products, additional primers for the VP1 region were redesigned for further genotyping of the remaining small portion non-EV71/non-CA16 specimens. With a known panel, it was possible to identify 15 out of 16 members using 5'-UTR sequence typing and VP1 typing, suggesting good detectability and genotyping of this method. One strain that was not typed by 5'-UTR was shown to be a recombinant virus. When this method was applied to examine clinical specimens from 44 suspected HFMD patients, 41 were detected as EV positive. In only one case, the VP1 sequence could not be identified. Four types of EVs, including CA16 (26/41, 63.4%), EV71-C4 (6/41, 14.6%), CA6 (5/41, 12.2%) and CA10 (3/41, 7.3%), were detected. In conclusion, 5' UTR amplification sequencing and subsequent VP1 specific primer amplification ensures a high detection rate and good genotyping accuracy in the examination of clinical samples. This detection strategy can be used for routine evaluation and monitoring of HFMD to follow local trends of EV infection.

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

    SciTech Connect

    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 dissolver

  6. Lactic acid jet test: in vitro erosion rates of glass ionomer dental cements containing radiopacifying elements.

    PubMed

    Williams, J A; Billington, R W; Pearson, G J

    1993-06-01

    The lactic acid jet test erosion rates were measured for 13 radiopaque glass ionomer dental materials obtained from a number of manufacturing sources. The erosion rate was compared with that found for the non-radiopaque restorative from the same manufacturer to determine whether the addition of an extra element had affected the resistance to erosion. Six materials were not significantly affected, six showed a significant increase in erosion rate. Only one material showed a reduced erosion rate. Materials containing a high proportion of any additive could show an increased erosion rate. Glass ionomer cements with or without radiopacifying elements had low erosion rates compared with other dental materials.

  7. Antibody and Viral Nucleic Acid Testing of Serum and Cerebrospinal Fluid for Diagnosis of Eastern Equine Encephalitis.

    PubMed

    Sherwood, James A; Brittain, David C; Howard, John J; Oliver, JoAnne

    2015-08-01

    Eastern equine encephalitis diagnostic serum antibody can appear 6 days after the onset of symptoms, and its numbers can increase 4-fold in 4 days, arguing for early and frequent serum testing. In populations where cerebrospinal fluid viral nucleic acid testing sensitivity and specificity remain undetermined, cerebrospinal antibody testing should also be performed.

  8. Simultaneous monitoring of glucose and uric acid on a single test strip with dual channels.

    PubMed

    Guo, Jinhong; Ma, Xing

    2017-03-15

    The conventional test strip has usually only one electrochemical reaction channel, which requires two times figure punctures for the self-management of patients suffering from both diabetes and gout. Considering the large number of such patients and for the sake of reducing their pains, we report an enzymatic test strip which can simultaneously monitor glucose and uric acid (UA) with only one fingertip blood droplet. The proposed test strip is composed of dual channels. The glucose in blood is detected in the 1st channel above on the substrate and the UA is characterized in the 2nd channel located at the bottom of the substrate. The proposed design intensively matches the requirement of those patients simultaneously suffering from diabetes and gout. We carried out comparative investigations on the proposed test strip and clinical biochemical analyser, which indicates a good agreement and proved the reliability and accuracy of the proposed test strip, as promising solution for the fast growth of family health management market.

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

    SciTech Connect

    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.

  10. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

    PubMed Central

    Roth, B L; Poot, M; Yue, S T; Millard, P J

    1997-01-01

    A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility. PMID:9172364

  11. Lipogenesis and lipid peroxidation in rat testes after long-term treatment with sucrose and tannic acid in drinking water.

    PubMed

    Mašek, T; Starčević, K

    2016-06-30

    We studied the influence of long-term treatment with sucrose and tannic acid in drinking water on the fatty acid profile and lipid peroxidation in rat testes. Male Wistar rats were supplemented with sucrose (30% w/v) or with sucrose and tannic acid (sucrose 30% w/v, tannic acid 0.1% w/v) in drinking water. The treatment with sucrose elevated blood glucose levels in the plasma (p < .05) and decreased the testis weight (p < .05) and testis index (p < .05) of the rats. Sucrose treatment increased monounsaturated fatty acids (MUFA) and C22:6n3, and decreased n6 fatty acids in testis tissue. Lipid peroxidation was significantly increased after sucrose administration in plasma (p < .05) and testis tissue (p < .01). The addition of tannic acid led to the decrease in lipid peroxidation in the plasma (p < .05) and testis (p < .05), a further increase in MUFA and decrease in n6 fatty acids. In conclusion, sucrose significantly altered the testis fatty acid profile with an increase in MUFA and C22:6n3, and a decrease in n6 fatty acids. Tannic acid attenuated oxidative stress and hyperglycaemia, but it did not improve pathological changes in the fatty acid composition of the testis.

  12. [The titration of double bonds in fatty acids of blood plasma in patients in testing of glucose tolerance].

    PubMed

    Titov, V N; Sazhina, N N; Evteeva, N M; Aripovskiĭ, A V; Tkhagalizhokova, E M

    2015-01-01

    The article deals with per oral glucose tolerance test applied to 20 patients with arterial hypertension. The blood plasma was analyzed to detect content of individual fatty acids, double bounds, glucose, insulin and metabolites of fatty acids. In patients with different resistance to insulin content of non-etherized fatty acids decreased approximatively up to 3 times. Without insulin resistance secretion of insulin in 2 hours after glucose load increased up to 3 times and content of individual fatty acids decreases in greater extent. Under insulin resistance secretion of insulin increases up to 8 times and decreasing of content of fatty acids is less expressed. The decrease in blood plasma of content of oleic and linoleic fatty acids and double bounds reflects effectiveness of effect of insulin--blockade of hydrolysis of triglycerides in subcutaneous adipocytes. The concentration of insulin positively correlates with initial content of palmitic fatty acid in the pool of lipids of blood plasma.

  13. Chemical Amplification with Encapsulated Reagents

    NASA Technical Reports Server (NTRS)

    Chen, Jian; Koemer, Steffi; Craig, Stephen; Lin, Shirley; Rudkevich, Dmitry M.; Rebek, Julius, Jr.

    2002-01-01

    Autocatalysis and chemical amplification are characteristic properties of living systems, and they give rise to behaviors such as increased sensitivity, responsiveness, and self-replication. Here we report a synthetic system in which a unique form of compartmentalization leads to nonlinear, autocatalytic behavior. The compartment is a reversibly formed capsule in which a reagent is sequestered. Reaction products displace the reagent from the capsule into solution and the reaction rate is accelerated. The resulting self-regulation is sensitive to the highly selective molecular recognition properties of the capsule.

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

    SciTech Connect

    Wiberg, Gustav K. H. E-mail: m.arenz@chem.ku.dk; Fleige, Michael; Arenz, Matthias E-mail: m.arenz@chem.ku.dk

    2015-02-15

    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 temperature, i.e., very close to the actual conditions in high temperature proton exchange membrane fuel cells (HT-PEMFCs). The cell consists of a stainless steel flow field and a PEEK plastic cell body comprising the electrochemical cell, which exhibits a three electrode configuration. The cell body and flow field are braced using a KF-25 vacuum flange clamp, which allows an easy assembly of the setup. As demonstrated, the setup can be used to investigate temperature dependent electrochemical processes on high surface area type electrocatalysts, but it also enables quick screening tests of HT-PEMFC catalysts under realistic conditions.

  15. Six-year pilot study on nucleic acid testing for blood donations in China.

    PubMed

    Ye, Xianlin; Yang, Baocheng; Zhu, Weigang; Zheng, Xin; Du, Peng; Zeng, Jingfeng; Li, Chengyao

    2013-10-01

    A six-year pilot study on nucleic acid testing for HBV, HCV and HIV-1 has been undertaken on sero-negative plasmas in mini-pool and individual donation testing at Shenzhen Blood Center. Of 307,740 sero-negative blood samples, 95 of 102 HBV DNA yields were confirmed positive, 80/95 (84.2%) were classified as occult HBV infection (OBI) and 15 (15.8%) as window period cases. Amongst OBIs, 45% carried anti-HBc only, 41.3% anti-HBc and anti-HBs and 13.7% anti-HBs only. HBV DNA yield was 1:3239. One HCV WP and one HIV-1 infected donations were detected. High residual risk was found in current blood donations screening in China.

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

    NASA Astrophysics Data System (ADS)

    Huang, Guoliang; Ma, Li; Yang, Xiaoyong; Yang, Xu

    2011-01-01

    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.

  17. Influence of pH on organic acid production by Clostridium sporogenes in test tube and fermentor cultures.

    PubMed Central

    Montville, T J; Parris, N; Conway, L K

    1985-01-01

    The influence of pH on the growth parameters of and the organic acids produced by Clostridium sporogenes 3121 cultured in test tubes and fermentors at 35 degrees C was examined. Specific growth rates in the fermentor maintained at a constant pH ranged from 0.20 h-1 at pH 5.00 to 0.86 h-1 at pH 6.50. Acetic acid was the primary organic acid in supernatants of 24-h cultures; total organic acid levels were 2.0 to 22.0 mumol/ml. Supernatants from pH 5.00 and 5.50 cultures had total organic acid levels less than one-third of those found at pH 6.00 to 7.00. The specific growth rates of the test tube cultures ranged from 0.51 h-1 at pH 5.00 to 0.95 h-1 at pH 6.50. The pH of the medium did not affect the average total organic acid content (51.5 mumol/ml) but did affect the distribution of the organic acids, which included formic, acetic, propionic, butyric, 3-(p-hydroxyphenyl)propionic, and 3-phenylpropionic acids. Butyric acid levels were lower, but formic and propionic acid levels were higher, at pH 5.00 than at other pHs. PMID:4004207

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

    SciTech Connect

    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.

  19. Agreement between the fatty acid ethyl ester hair test for alcohol and social workers' reports.

    PubMed

    Kulaga, Vivian; Gareri, Joey; Fulga, Netta; Koren, Gideon

    2010-06-01

    The purpose of this study was to examine the relationship between social worker reports and the fatty acid ethyl ester (FAEE) test as a biomarker for heavy alcohol use. In 2005, a diagnostic program to detect excessive alcohol use by FAEE hair analysis in parents at high risk of having children with fetal alcohol spectrum disorders was established. All cases submitted by Child Protective Services between May and December of 2007 (n = 172) were included comparing social worker reports with FAEE test outcome by odds ratio analysis. A subanalysis of mothers (n = 119), excluding fathers, was also performed. Factors associated with testing positive for hair FAEE in parents, and mothers alone, were: knowledge of a specific instance of problem drinking within the past 6 months (odds ratio [OR] = 5.11, 2.57-10.16 and OR = 8.51, 3.59-20.18, respectively) and third party reports alleging alcohol abuse (OR = 3.31, 1.69-6.46 and OR = 3.30, 1.45-7.50, respectively). Mothers who admitted to heavy drinking were also seven times more likely to test positive for hair FAEE (OR = 6.74, 1.50-30.38) than those who did not. Factors negatively associated with testing positive for hair FAEE in parents, and mothers alone, were: social workers testing for FAEE without the suspicion of alcohol use but rather as a measure to "cover all bases" (OR = 0.09, 0.02-0.40 and (OR = 0.13, 0.03-0.58, respectively) or because of a history/suspicion of illicit drug use (OR = 0.2, 0.07-0.55 and OR = 0.26, 0.08-0.80, respectively). Eleven of 15 reports, indicating levels of consumption, were also in clinical agreement with FAEE test outcome. The FAEE hair test is being applied for the first time in the present context. Our results show the test corroborates well with social workers' suspicion of alcohol use. Reported factors directly related to alcohol use were significantly associated with testing positive for excessive alcohol use, whereas factors not directly related to alcohol use were negatively

  20. Trophic amplification of climate warming.

    PubMed

    Kirby, Richard R; Beaugrand, Gregory

    2009-12-07

    Ecosystems can alternate suddenly between contrasting persistent states due to internal processes or external drivers. It is important to understand the mechanisms by which these shifts occur, especially in exploited ecosystems. There have been several abrupt marine ecosystem shifts attributed either to fishing, recent climate change or a combination of these two drivers. We show that temperature has been an important driver of the trophodynamics of the North Sea, a heavily fished marine ecosystem, for nearly 50 years and that a recent pronounced change in temperature established a new ecosystem dynamic regime through a series of internal mechanisms. Using an end-to-end ecosystem approach that included primary producers, primary, secondary and tertiary consumers, and detritivores, we found that temperature modified the relationships among species through nonlinearities in the ecosystem involving ecological thresholds and trophic amplifications. Trophic amplification provides an alternative mechanism to positive feedback to drive an ecosystem towards a new dynamic regime, which in this case favours jellyfish in the plankton and decapods and detritivores in the benthos. Although overfishing is often held responsible for marine ecosystem degeneration, temperature can clearly bring about similar effects. Our results are relevant to ecosystem-based fisheries management (EBFM), seen as the way forward to manage exploited marine ecosystems.

  1. Trophic amplification of climate warming

    PubMed Central

    Kirby, Richard R.; Beaugrand, Gregory

    2009-01-01

    Ecosystems can alternate suddenly between contrasting persistent states due to internal processes or external drivers. It is important to understand the mechanisms by which these shifts occur, especially in exploited ecosystems. There have been several abrupt marine ecosystem shifts attributed either to fishing, recent climate change or a combination of these two drivers. We show that temperature has been an important driver of the trophodynamics of the North Sea, a heavily fished marine ecosystem, for nearly 50 years and that a recent pronounced change in temperature established a new ecosystem dynamic regime through a series of internal mechanisms. Using an end-to-end ecosystem approach that included primary producers, primary, secondary and tertiary consumers, and detritivores, we found that temperature modified the relationships among species through nonlinearities in the ecosystem involving ecological thresholds and trophic amplifications. Trophic amplification provides an alternative mechanism to positive feedback to drive an ecosystem towards a new dynamic regime, which in this case favours jellyfish in the plankton and decapods and detritivores in the benthos. Although overfishing is often held responsible for marine ecosystem degeneration, temperature can clearly bring about similar effects. Our results are relevant to ecosystem-based fisheries management (EBFM), seen as the way forward to manage exploited marine ecosystems. PMID:19740882

  2. Dynamics and control of DNA sequence amplification

    NASA Astrophysics Data System (ADS)

    Marimuthu, Karthikeyan; Chakrabarti, Raj

    2014-10-01

    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.

  3. Dynamics and control of DNA sequence amplification

    SciTech Connect

    Marimuthu, Karthikeyan; Chakrabarti, Raj E-mail: rajc@andrew.cmu.edu

    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.

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

    SciTech Connect

    J. Francfort; J. Argueta; M. Wehrey; D. Karner; L. Tyree

    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.

  5. Accelerated cycle-life testing of small sealed lead/acid batteries

    NASA Astrophysics Data System (ADS)

    Kim, I.; Oh, S. H.; Kang, H. Y.

    An attempt has been made to devise methods for reducing the cycle-testing time of long-life sealed lead/acid batteries. In order for the accelerated test results to equate to the actual field operations, it is assumed that the failure modes under both normal and accelerated conditions must be the same. As a first step in the search for a reliable accelerated test, observations of the battery ageing process have been made under different daily duty cycles, viz., 1 (normal), 8 and 16 cycles/day at ambient temperature and 80% depth-of-discharge. It has been found that the main cause of failure is different for a given duty cycle. This complicates the task of applying accelerated test results to field operations. For the 8 cycles/day schedule, the main cause of failure is degradation of the positive active material. Positive grid corrosion is the main factor in the 16 cycles/day case. Under normal conditions, both grid corrosion and PbO 2 degradation appear to be equally significant.

  6. Small Sample Whole-Genome Amplification

    SciTech Connect

    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.

  7. Small sample whole-genome amplification

    NASA Astrophysics Data System (ADS)

    Hara, Christine; Nguyen, Christine; Wheeler, Elizabeth; Sorensen, Karen; Arroyo, Erin; Vrankovich, Greg; Christian, Allen

    2005-11-01

    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.

  8. Isothermal amplification coupled with rapid flow-through hybridisation for sensitive diagnosis of Plum pox virus.

    PubMed

    Olmos, Antonio; Bertolini, Edson; Cambra, Mariano

    2007-01-01

    A nucleic acid sequence-based amplification method coupled with rapid flow-through hybridisation (NASBA-FH) was developed for diagnosis of Plum pox virus (PPV). The sensitivity level achieved by NASBA-FH was 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 262 stone-fruit trees collected during winter and spring seasons were analysed. These samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. Winter PPV diagnostic results by ELISA and NASBA-FH coincided in 90.8%, while ELISA and PCR-based methods coincided in 91.6% and PCR-based methods with NASBA-FH agreed in 95.4%. In spring, diagnostic results were similar with all the molecular techniques, which agreed with ELISA results for 98.8% of the trees. NASBA-FH was able to detect more positive infections in winter, which were later confirmed in spring. These results indicate that NASBA-FH is a suitable molecular method for routine PPV detection in the winter and spring. This user-friendly isothermal RNA amplification coupled with a very fast flow-through hybridisation (15 min) opens up new possibilities for rapid and reliable diagnosis of a variety of pathogens.

  9. Basin amplification of seismic waves in the city of Pahrump, Nevada.

    SciTech Connect

    Abbott, Robert E.

    2005-07-01

    Sedimentary basins can increase the magnitude and extend the duration of seismic shaking. This potential for seismic amplification is investigated for Pahrump Valley, Nevada-California. The Pahrump Valley is located approximately 50 km northwest of Las Vegas and 75 km south of the Nevada Test Site. Gravity data suggest that the city of Pahrump sits atop a narrow, approximately 5 km deep sub-basin within the valley. The seismic amplification, or ''site effect'', was investigated using a combination of in situ velocity modeling and comparison of the waveforms and spectra of weak ground motion recorded in the city of Pahrump, Nevada, and those recorded in the nearby mountains. Resulting spectral ratios indicate seismic amplification factors of 3-6 over the deepest portion of Pahrump Valley. This amplification predominantly occurs at 2-2.5 Hz. Amplification over the deep sub-basin is lower than amplification at the sub-basin edge, location of the John Blume and Associates PAHA seismic station, which recorded many underground nuclear tests at the Nevada Test Site. A comprehensive analysis of basin amplification for the city of Pahrump should include 3-D basin modeling, due to the extreme basement topography of the Pahrump Valley.

  10. Comparative evaluation of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

    PubMed Central

    Piersimoni, C; Callegaro, A; Nista, D; Bornigia, S; De Conti, F; Santini, G; De Sio, G

    1997-01-01

    Two commercial assays detecting the presence of Mycobacterium tuberculosis complex in clinical specimens by rRNA target amplification (Gen-Probe Amplified M. tuberculosis Direct Test [AMTD]) and PCR (Amplicor) were evaluated. The tests were applied to 327 digested, decontaminated respiratory specimens collected from 236 patients. Results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered the "gold standard." A total of 60 specimens were collected from 27 patients with a diagnosis of pulmonary tuberculosis. Thirteen of these specimens were from patients receiving standard antituberculosis therapy and therefore were not included in the comparison. Of the remaining 47 specimens, 33 were smear positive, 40 were culture positive, 45 were AMTD positive, and 39 were Amplicor positive. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values were 77, 100, 100, and 95 for staining; 87, 100, 100, and 97.4 for culture; 95.9, 98.9, 94, and 99.2 for AMTD; and 85.4, 99.6, 97.9, and 97.1 for Amplicor, respectively. Agreement between AMTD and Amplicor assay results was 96.8%. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection of M. tuberculosis complex in respiratory specimens, AMTD appeared to be more sensitive than Amplicor. PMID:8968906

  11. Droplet microfluidics for amplification-free genetic detection of single cells.

    PubMed

    Rane, Tushar D; Zec, Helena C; Puleo, Chris; Lee, Abraham P; Wang, Tza-Huei

    2012-09-21

    In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.

  12. Development of loop-mediated isothermal amplification assay for detection of human coronavirus-NL63.

    PubMed

    Pyrc, Krzysztof; Milewska, Aleksandra; Potempa, Jan

    2011-07-01

    Human coronavirus NL63 was identified in 2004 in the Netherlands. Due to the high prevalence and world-wide distribution of this pathogen, it is essential to develop a sensitive and specific detection assay suitable for use in a routine diagnostic laboratory. Techniques based on PCR or real-time PCR are laborious and expensive. Detailed analysis of the HCoV-NL63 genome permitted the identification of a conserved nucleic acid sequential motif, which was sufficient for the design of a loop-mediated isothermal amplification (LAMP) assay. Evaluation of the method showed that the test is specific to HCoV-NL63 and that it does not cross-react with other respiratory viruses. The detection limit was found to be 1 copy of RNA template per reaction in cell culture supernatants and clinical specimens.

  13. Chirality Amplification in Tactoids of Lyotropic Chromonic Liquid Crystals

    NASA Astrophysics Data System (ADS)

    Peng, Chenhui; Lavrentovich, Oleg

    2014-03-01

    We demonstrate an effective chirality amplification based on the long-range forces, extending over the scales of tens of micrometers, much larger than the single molecule (nanometer) scale. The mechanism is rooted in the long-range elastic nature of orientational order in lyotropic chromonic liquid crystals (LCLCs) that represent water solutions of achiral disc-like molecules. Minute quantities of chiral molecules such as amino acid L-alanine and limonene added to the droplets of LCLC lead to chiral amplification characterized by an increase of optical activity by a factor of 103 - 104. This effect allows one to discriminate and detect the absolute configuration of chiral molecules in an aqueous system, thus opening new possibilities in biosensing and other biological applications.

  14. Geometric Effects on the Amplification of First Mode Instability Waves

    NASA Technical Reports Server (NTRS)

    Kirk, Lindsay C.; Candler, Graham V.

    2013-01-01

    The effects of geometric changes on the amplification of first mode instability waves in an external supersonic boundary layer were investigated using numerical techniques. Boundary layer stability was analyzed at Mach 6 conditions similar to freestream conditions obtained in quiet ground test facilities so that results obtained in this study may be applied to future test article design to measure first mode instability waves. The DAKOTA optimization software package was used to optimize an axisymmetric geometry to maximize the amplification of the waves at first mode frequencies as computed by the 2D STABL hypersonic boundary layer stability analysis tool. First, geometric parameters such as nose radius, cone half angle, vehicle length, and surface curvature were examined separately to determine the individual effects on the first mode amplification. Finally, all geometric parameters were allowed to vary to produce a shape optimized to maximize the amplification of first mode instability waves while minimizing the amplification of second mode instability waves. Since first mode waves are known to be most unstable in the form of oblique wave, the geometries were optimized using a broad range of wave frequencies as well as a wide range of oblique wave angles to determine the geometry that most amplifies the first mode waves. Since first mode waves are seen most often in flows with low Mach numbers at the edge of the boundary layer, the edge Mach number for each geometry was recorded to determine any relationship between edge Mach number and the stability of first mode waves. Results indicate that an axisymmetric cone with a sharp nose and a slight flare at the aft end under the Mach 6 freestream conditions used here will lower the Mach number at the edge of the boundary layer to less than 4, and the corresponding stability analysis showed maximum first mode N factors of 3.

  15. Transmission of Hepatitis C Virus From Organ Donors Despite Nucleic Acid Test Screening.

    PubMed

    Suryaprasad, A; Basavaraju, S V; Hocevar, S N; Theodoropoulos, N; Zuckerman, R A; Hayden, T; Forbi, J C; Pegues, D; Levine, M; Martin, S I; Kuehnert, M J; Blumberg, E A

    2015-07-01

    Nucleic acid testing (NAT) for hepatitis C virus (HCV) is recommended for screening of organ donors, yet not all donor infections may be detected. We describe three US clusters of HCV transmission from donors at increased risk for HCV infection. Donor's and recipients' medical records were reviewed. Newly infected recipients were interviewed. Donor-derived HCV infection was considered when infection was newly detected after transplantation in recipients of organs from increased risk donors. Stored donor sera and tissue samples were tested for HCV RNA with high-sensitivity quantitative PCR. Posttransplant and pretransplant recipient sera were tested for HCV RNA. Quasispecies analysis of hypervariable region-1 was used to establish genetic relatedness of recipient HCV variants. Each donor had evidence of injection drug use preceding death. Of 12 recipients, 8 were HCV-infected-6 were newly diagnosed posttransplant. HCV RNA was retrospectively detected in stored samples from donor immunologic tissue collected at organ procurement. Phylogenetic analysis showed two clusters of closely related HCV variants from recipients. These investigations identified the first known HCV transmissions from increased risk organ donors with negative NAT screening, indicating very recent donor infection. Recipient informed consent and posttransplant screening for blood-borne pathogens are essential when considering increased risk donors.

  16. Identification of staphylococci with a self-educating system using fatty acid analysis and biochemical tests.

    PubMed Central

    Behme, R J; Shuttleworth, R; McNabb, A; Colby, W D

    1996-01-01

    We characterized all of the 35 aerobic taxa of the genus Staphylococcus by using an objective, self-learning system combining both whole-cell fatty acid (FA) analysis and the results of 35 biochemical tests. Isolates were compared with the type strain for each taxon to generate an FA profile library and a biochemical table of test responses. Isolates were accepted into the system if they had a similarity index of > or = 0.6 for a taxon within the FA profile library and if they were identified as the same taxon by a computer program using a probability matrix constructed from the biochemical data. These stringent criteria led to acceptance of 1,117 strains assigned to legitimate taxa. Additional FA groups were assembled from selected strains that did not meet the inclusion criteria based on the type strains and were added to the system as separate entries. Currently, 1,512 isolates have bee accepted into the system. This approach has resulted in a comprehensive table of biochemical test results and a FA profile library, which together provide a practical system for valid identifications. PMID:8940451

  17. Changes in the serum composition of free-fatty acids during an intravenous glucose tolerance test.

    PubMed

    Soriguer, Federico; García-Serrano, Sara; García-Almeida, Jose M; Garrido-Sánchez, Lourdes; García-Arnés, Juan; Tinahones, Francisco J; Cardona, Isabel; Rivas-Marín, Jose; Gallego-Perales, Jose L; García-Fuentes, Eduardo

    2009-01-01

    Recent studies suggest that measuring the free-fatty acids (FFA) during an intravenous glucose tolerance test (IVGTT) may provide information about the metabolic associations between serum FFA and carbohydrate and insulin metabolism. We evaluated the FFA profile during an IVGTT and determined whether this test changes the composition and concentration of FFA. An IVGTT was given to 38 severely obese persons before and 7 months after undergoing bariatric surgery and also to 12 healthy, nonobese persons. The concentration and composition of the FFA were studied at different times during the test. The concentration of FFA fell significantly faster during the IVGTT in the controls and in the severely obese persons with normal-fasting glucose (NFG) than in the severely obese persons with impaired-fasting glucose (IFG) or type 2 diabetes mellitus (T2DM) (P < 0.05). Significant differences were found in the time to minimum serum concentrations of FFA (control = NFG < IFG < T2DM) (P < 0.001). These variables improved after bariatric surgery in the three groups. The percentage of monounsaturated and n-6 polyunsaturated FFA in the control subjects and in the obese persons, both before and after surgery, decreased significantly during the IVGTT. In conclusion, during an IVGTT, severely obese persons with IFG or T2DM experienced a lower fall in the FFA than the severely obese persons with NFG and the controls, becoming normal after bariatric surgery.

  18. Multiscale image contrast amplification (MUSICA)

    NASA Astrophysics Data System (ADS)

    Vuylsteke, Pieter; Schoeters, Emile P.

    1994-05-01

    This article presents a novel approach to the problem of detail contrast enhancement, based on multiresolution representation of the original image. The image is decomposed into a weighted sum of smooth, localized, 2D basis functions at multiple scales. Each transform coefficient represents the amount of local detail at some specific scale and at a specific position in the image. Detail contrast is enhanced by non-linear amplification of the transform coefficients. An inverse transform is then applied to the modified coefficients. This yields a uniformly contrast- enhanced image without artefacts. The MUSICA-algorithm is being applied routinely to computed radiography images of chest, skull, spine, shoulder, pelvis, extremities, and abdomen examinations, with excellent acceptance. It is useful for a wide range of applications in the medical, graphical, and industrial area.

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

    PubMed

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

    2010-04-01

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

  20. Third Sound Amplification and Detailed Balance

    SciTech Connect

    Eddinger, J. D.; Ellis, F. M.

    2006-09-07

    Condensation of atoms from the vapor into a third sound resonance is expected to be capable of acoustic amplification. This results from normal to superfluid conversion that coherently accommodates atoms into the third sound velocity field. Consideration of third sound in light of the equilibrium detailed balance between vapor particles and the superfluid film provides further evidence that acoustic amplification is attainable.

  1. Classroom Amplification Technology: Theory and Practice.

    ERIC Educational Resources Information Center

    Crandell, Carl C.; Smaldino, Joseph J.

    2000-01-01

    This article reviews some relevant events in the development of acoustical standards for classrooms, describes classroom challenges to providing clear acoustical signals to children in classrooms, and outlines amplification solutions to some of those classroom challenges. Solutions include personal amplification devices and use of signal-to-noise…

  2. 21 CFR 862.1055 - Newborn screening test system for amino acids, free carnitine, and acylcarnitines using tandem...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... mass spectrometry is a device that consists of stable isotope internal standards, control materials... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Newborn screening test system for amino acids... CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1055...

  3. 78 FR 63476 - Draft Guidance for Industry: Use of Nucleic Acid Tests To Reduce the Risk of Transmission of West...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-24

    ... Reduce the Risk of Transmission of West Nile Virus From Donors of Human Cells, Tissues, and Cellular and... ``Guidance for Industry: Use of Nucleic Acid Tests to Reduce the Risk of Transmission of West Nile Virus From... donors of HCT/Ps, with recommendations for donor testing for West Nile Virus (WNV) using an...

  4. Underestimation of pyruvic acid concentrations by fructose and cysteine in 2,4-dinitrophenylhydrazine-mediated onion pungency test.

    PubMed

    Yoo, Kil Sun; Lee, Eun Jin; Patil, Bhimanagouda S

    2011-10-01

    Onion pungency has been routinely measured by determining pyruvic acid concentration in onion juice by reacting with 2,4-dinitrophenylhydrazine (DNPH) since 1961. However, the absorbency of the color adduct of the reaction rapidly decreased in onion samples as compared to that of the pyruvic acid standards, resulting in underestimations of the pyruvic acid concentrations. By measuring the absorbency at 1 min, we have demonstrated that accuracy could be substantially improved. As a continuation, the causes of degradation of the color adduct after the reaction and pyruvic acid itself before the reaction were examined in this study. Alliinase action in juice (fresh or cooked) and bulb colors did not influence the degradation. Some organic acids indigenously found in onion, such as ascorbic acid, proline, and glutamic acid, did not reduce the absorbency. However, fructose within the onion juice or supplemented caused the degradation of the color adduct, whereas sucrose and glucose had a lesser effect. Degradation rates increased proportionally as fructose concentrations increased up to 70 mg/mL. Cysteine was found to degrade the pyruvic acid itself before the pyruvic acid could react with DNPH. Approximately 90% of the pyruvic acid was degraded after 60 min in samples of 7 mM pyruvic acid supplemented with 10 mg/mL cysteine. Spectral comparisons of onion juice containing fructose naturally and pyruvic acid solution with supplemented fructose indicated identical patterns and confirmed that the color-adduct degradation was caused by fructose. Our study elucidated that fructose, a major sugar in onion juice, caused the degradation of color adduct in the onion pungency test and resulted in underestimation of the pyruvic acid concentration.

  5. [The content of individual fatty acids and numbers of double bonds, insulin, C-peptide and unesterified fatty acids in blood plasma in testing tolerance to glucose].

    PubMed

    Titov, V N; Sazhina, N N; Aripovskiĭ, A V; Evteeva, N M; Tkhagalizhokova, É M; Parkhimovich, R M

    2014-10-01

    The glucose tolerance test demonstrates that content of unesterified fatty acids in blood plasma decreases up to three times and the content of oleic and linoleic acids is more decreased in the pool of fatty acids lipids. Out of resistance to insulin, hormone secretion increases up to three times. The decreasing of level of individual fatty acids occurs in a larger extent. Under resistance to insulin secretion of insulin is increasing up to eight times. The decreasing of level of each fatty acid is less expressed. The effect of insulin reflects decreasing of content of double bonds in blood plasma. The number of double bonds characterizes the degree of unsaturation of fatty acids in lipids of blood plasma. The higher number of double bonds is in the pool of unesterified fatty acids the more active is the effect of insulin. The hyper-secretion of insulin is directly proportional to content of palmitic fatty acid in lipids of blood plasma on fasting. According the phylogenetic theory of general pathology, the effect of insulin on metabolism of glucose is mediated by fatty acids. The insulin is blocking lipolysis in insulin-depended subcutaneous adipocytes and decreases content of unesterified fatty acids in blood plasma. The insulin is depriving all cells of possibility to absorb unesterified fatty acids and "forces" them to absorb glucose increasing hereby number of GLUT4 on cell membrane. The resistance to insulin is manifested in high concentration of unesterfied fatty acids, hyperinsulinemia, hyperalbuminemia and increasing of concentration of C-reactive protein-monomer. The resistance to insulin is groundlessly referred to as a symptom of diabetes mellitus type II. The resistance to insulin is only a functional disorder lasting for years. It can be successfully arrested. The diabetes mellitus is developed against the background of resistance to insulin only after long-term hyper-secretion of insulin and under emaciation and death of β-cells. The diabetes

  6. Optimization of a phage amplification assay to permit accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis cells.

    PubMed

    Foddai, Antonio; Elliott, Christopher T; Grant, Irene R

    2009-06-01

    A commercially available phage amplification assay, FASTPlaqueTB (Biotec Laboratories, Ipswich, United Kingdom), when used according to the manufacturer's instructions, does not permit accurate enumeration of Mycobacterium avium subsp. paratuberculosis. The aim of this study was to optimize the phage amplification assay conditions to permit accurate quantification of viable M. avium subsp. paratuberculosis cells. The burst time for M. avium subsp. paratuberculosis was initially determined to inform decisions about optimal incubation time before plating, and then other test parameters were altered to evaluate how the correlation between plaque and colony counts was affected. The D29 mycobacteriophage replicates more slowly in M. avium subsp. paratuberculosis than in Mycobacterium smegmatis (used to optimize the commercial test originally), and the mean burst time for four M. avium subsp. paratuberulosis strains was 210 +/- 36.8 min at 37 degrees C compared to 63 +/- 17.5 min for M. smegmatis mc(2) 155. To achieve 100% correlation between plaque and colony counts, the optimized phage assay includes the following: (i) resuspension of the samples to be tested in Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase and 2 mM calcium chloride, followed by overnight incubation at 37 degrees C before performance of the phage assay; (ii) a 2-h incubation of the sample with D29 mycobacteriophage before viricide treatment; and (iii) a further 90-min incubation after viricide treatment and neutralization up to the burst time (total incubation time, 210 min) before plating with M. smegmatis mc(2) 155 in 7H9 agar. The optimized phage amplification assay was able to detect 1 to 10 CFU/ml of M. avium subsp. paratuberculosis in spiked milk or broth within 48 h, as demonstrated by the results of several blind trials.

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

    PubMed

    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.

  8. Use of a novel metal indicator to judge loop-mediated isothermal amplification for detecting the 35S promoter.

    PubMed

    Wang, Xiaofu; Fu, Zhenfang; Chen, Xiaoyun; Peng, Cheng; Xu, Xiaoli; Wei, Wei; Li, Feiwu; Xu, Junfeng

    2017-02-01

    Loop-mediated isothermal amplification (LAMP) is a widely used isothermal nucleic acid amplification method. Here we developed a new closed-tube colorimetric method for judging LAMP with a novel metal indicator. First, the metal indicator, acid chrome blue K (ACBK), was evaluated in the LAMP reaction with various combinations of reaction reagents, such as reaction buffer, dNTP mixtures, primer mixtures, or Mg(2+). We found that the solution color of the LAMP reaction with ACBK changed from red to blue based on a decrease in the Mg(2+) concentration in the reaction solution. We then optimized the LAMP with ACBK method for detecting the Cauliflower Mosaic Virus 35S promoter. Further, the specificity of the new colorimetric assay using ACBK in the LAMP reaction for detecting the 35S promoter was tested with diverse transgenic events in different crops, and the sensitivity threshold of the assay was ∼50 copies for transgenic rice genomic DNA and 100 ng of 0.1 % DNA from rice, soybean, rapeseed, and maize. Finally, the applicability of the LAMP assay was successfully validated using practical maize samples. All the detection results could be easily discerned either by UV-vis spectroscopy or the naked eye. Graphical Abstract The visual detect LAMP amplification by the addition of ACBK as a signal indicator. The color of the LAMP-ACBK solution turned from red to blue as the concentration of free Mg(2+) decreases. The detection results could be easily discerned either by UV-vis spectroscopy or the naked eye.

  9. HER-2/neu and topoisomerase IIa gene amplification and protein expression in invasive breast carcinomas: chromogenic in situ hybridization and immunohistochemical analyses.

    PubMed

    Bhargava, Rohit; Lal, Priti; Chen, Beiyun

    2005-06-01

    We studied HER-2/neu (HER-2) and topoisomerase IIa (topo2a) amplification (using chromogenic in situ hybridization) and overexpression (immunohistochemical analysis) in 113 invasive breast carcinomas. A gene copy number/chromosome 17 copy number ratio of 2.0 or higher indicated amplification. A topo2a/chromosome 17 ratio of less than 0.8 indicated gene deletion. HER-2 overexpression was scored according to standard HercepTest guidelines (DAKO, Carpinteria, CA). Overexpression of topo2a was identified when nuclear staining was found in more than 5% of tumor cells. Of 113 tumors, 104 were analyzed successfully for HER-2 and topo2a amplification. Of the 104, 64 showed HER-2 amplification; 25 of these (39%) also showed topo2a amplification. No amplification was found in 40 tumors. Deletion of topo2a was seen in 7 (11%) of 64 HER-2-amplified tumors and 2 (5%) of 40 nonamplified tumors. Of 25 tumors with topo2a amplification, 18 (72%) overexpressed topo2a. Only 3 (4%) of 79 tumors without topo2a amplification overexpressed topo2a. Amplification of topo2a is associated with HER-2 amplification but not vice versa. Amplification of topo2a resulted in protein overexpression in 72% of tumors, but topo2a overexpression rarely occurred without gene amplification. Identification of topo2a and HER-2 status might have therapeutic and prognostic implications.

  10. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

    PubMed

    Lucchi, Naomi W; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.

  11. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites

    PubMed Central

    Lucchi, Naomi W.; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

  12. Assessment of high power HEV lead-acid battery advancements by comparative benchmarking with a European test procedure

    NASA Astrophysics Data System (ADS)

    Conte, Mario; Pede, Giovanni; Sglavo, Vincenzo; Macerata, Diego

    The technical and practical suitability of lead-acid batteries for applications in vehicles with electrical drivetrains (battery-powered or hybrid electric) has been experimentally investigated in a variety of testing programmes. Under the direction and funding support of the Commission of the European Community, since early 1990s, the R&D Organisation EUCAR, a collaborative partnership of most European car manufacturers, has been conducting battery technological assessment projects, through bench tests carried out by different independent laboratories throughout Europe, using agreed test procedures. In this framework, ENEA acted as independent testing institute and tested, among others, three high power lead-acid batteries of various technologies (flat plate electrodes and spiral wound) for EV and HEV applications. In addition, different battery sizes and operating conditions have been tested at ENEA in a separate collaboration with ALTRA-IRISBUS. This paper intends to trace technological and performance improvements of high power lead-acid battery technology through the analysis of experimental data during parameter and life cycle tests, including the effects of battery sizes, charge/discharge profiles and testing procedures, with special emphasis on the reduction of the internal resistance and the variation of peak power and cycle life.

  13. Genotoxicity testing of three monohaloacetic acids in TK6 cells using the cytokinesis-block micronucleus assay.

    PubMed

    Liviac, Danae; Creus, Amadeu; Marcos, Ricard

    2010-09-01

    Chemical disinfection of water generates harmful chemical compounds, known as disinfection by-products (DBPs). One class of DBPs is constituted by haloacetic acids (HAAs), the second major group in prevalence (after trihalomethanes) detected in finished drinking water. In this article, we report the results obtained in the evaluation of the chromosome damage induced by three monohaloacetic acids, namely iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA). To evaluate the induction of chromosome damage, we used the cytokinesis-block micronucleus test that measures the ability of genotoxic agents to induce both clastogenic and/or aneugenic effects. No previous data exist on the effects of these compounds on human chromosomes. We tested five doses of each HAA, in addition to the negative and positive controls. The highest dose tested for each HAA was that immediately lower than the dose producing total cytotoxicity. Our results show that none of the three HAAs tested was able to increase significantly the frequency of micronucleus in binucleated TK6 cells, the rank order in decreasing cytotoxicity was IAA > BAA > CAA.

  14. How Alterations in the Cdt1 Expression Lead to Gene Amplification in Breast Cancer

    DTIC Science & Technology

    2011-07-01

    Charlottesville, VA 22904 kt5m@ri.ncvc.go.jp We examined the effects of Cdt1 for gene amplification by using inducible system . We Obtained and validated...reagents for inducible Cdt1-expression- system . Furthermore, we identified appropriated dose of MTX for testing gene amplification Cdt2 is a key...Doxcycline-inducible Cdt1-expression- system has reported by using Doxcycline (Liontos, Koutsami et al., 2007). We obtained these cell lines and

  15. Preferential Amplification of Pathogenic Sequences.

    PubMed

    Ge, Fang; Parker, Jayme; Chul Choi, Sang; Layer, Mark; Ross, Katherine; Jilly, Bernard; Chen, Jack

    2015-06-11

    The application of next generation sequencing (NGS) technology in the diagnosis of human pathogens is hindered by the fact that pathogenic sequences, especially viral, are often scarce in human clinical specimens. This known disproportion leads to the requirement of subsequent deep sequencing and extensive bioinformatics analysis. Here we report a method we called "Preferential Amplification of Pathogenic Sequences (PATHseq)" that can be used to greatly enrich pathogenic sequences. Using a computer program, we developed 8-, 9-, and 10-mer oligonucleotides called "non-human primers" that do not match the most abundant human transcripts, but instead selectively match transcripts of human pathogens. Instead of using random primers in the construction of cDNA libraries, the PATHseq method recruits these short non-human primers, which in turn, preferentially amplifies non-human, presumably pathogenic sequences. Using this method, we were able to enrich pathogenic sequences up to 200-fold in the final sequencing library. This method does not require prior knowledge of the pathogen or assumption of the infection; therefore, it provides a fast and sequence-independent approach for detection and identification of human viruses and other pathogens. The PATHseq method, coupled with NGS technology, can be broadly used in identification of known human pathogens and discovery of new pathogens.

  16. Toughness amplification in natural composites

    NASA Astrophysics Data System (ADS)

    Barthelat, Francois; Rabiei, Reza

    2011-04-01

    Natural structural materials such as bone and seashells are made of relatively weak building blocks, yet they exhibit remarkable combinations of stiffness, strength and toughness. This performance can be largely explained by their "staggered microstructure": stiff inclusions of high aspect ratio are laid parallel to each other with some overlap, and bonded by a softer matrix. While stiffness and strength are now well understood for staggered composites, the mechanisms involved in fracture are still largely unknown. This is a significant lack since the amplification of toughness with respect to their components is by far the most impressive feature in natural staggered composites such as nacre or bone. Here a model capturing the salient mechanisms involved in the cracking of a staggered structure is presented. We show that the pullout of inclusions and large process zones lead to tremendous toughness by far exceeding that of individual components. The model also suggests that a material like nacre cannot reach steady state cracking, with the implication that the toughness increases indefinitely with crack advance. These findings agree well with existing fracture data, and for the first time relate microstructural parameters with overall toughness. These insights will prove useful in the design of biomimetic materials, and provide clues on how bone fractures at the nano and microscales.

  17. Adipic acid enhanced flue gas desulfurization process for industrial boilers: Volume 1. Field test results. Project summary

    SciTech Connect

    Clarke, P.A.; Gerstle, R.W.; Henzel, D.S.; Mason, K.W.; Sabatini, S.R.

    1983-03-01

    Test results show that adding adipic acid to the limestone slurry significantly improved the SO/sub 2/ removal efficiency of the FGD system. Limited baseline data on operations with limestone only indicated a performance level of 55% SO/sub 2/ removal. Adding about 2200 ppM of adipic acid to the limestone scrubbing systems, the unit's level of performance increased to an average of 94.3% SO/sub 2/ removal which was maintained within a standard deviation of 2.2% over a 30-day test period during which boiler load was 70 to 130 million Btu/hr and gas throughput varied 300%.

  18. Folic acid absorption determined by a single stool sample test--a double-isotope technique. The folic acid absorption capacity in children

    SciTech Connect

    Hjelt, K. )

    1989-10-01

    The fractional folic acid absorption (FAFol) was determined in 66 patients with various gastrointestinal diseases by a double-isotope technique, employing a single stool sample test (SSST) as well as a complete stool collection. The age of the patients ranged from 2.5 months to 16.8 years (mean 6.3 years). The test dose was administered orally and consisted of 50 micrograms of (3H)folic acid (monoglutamate) (approximately 20 muCi), carmine powder, and 2 mg 51CrCl3 (approximately 1.25 muCi) as the unabsorbable tracer. The whole-body radiation given to a 1-year-old child averaged 4.8 mrad only. The stool and napkin contents were collected and homogenized by the addition of 300 ml chromium sulfuric acid. A 300-ml sample of the homogenized stool and napkin contents, as well as 300 ml chromium sulfuric acid (75% vol/vol) containing the standards, were counted for the content of 51Cr in a broad-based well counter. The quantity of (3H)folic acid was determined by liquid scintillation, after duplicate distillation. Estimated by SSST, the FAFol, which employs the stool with the highest content of 51Cr corresponding to the most carmine-colored stool, correlated closely with the FAFol based on complete stool collection (r = 0.96, n = 39, p less than 0.0001). The reproducibility of FAFol determined by SSST was assessed from repeated tests in 18 patients. For a mean of 81%, the SD was 4.6%, which corresponded to a coefficient of variation of 5.7%.

  19. Microsatellites Cross-Species Amplification across Some African Cichlids

    PubMed Central

    Bezault, Etienne; Rognon, Xavier; Gharbi, Karim; Baroiller, Jean-Francois; Chevassus, Bernard

    2012-01-01

    The transfer of the genomic resources developed in the Nile tilapia, Oreochromis niloticus, to other Tilapiines sensu lato and African cichlid would provide new possibilities to study this amazing group from genetics, ecology, evolution, aquaculture, and conservation point of view. We tested the cross-species amplification of 32 O. niloticus microsatellite markers in a panel of 15 species from 5 different African cichlid tribes: Oreochromines (Oreochromis, Sarotherodon), Boreotilapiines (Tilapia), Chromidotilapines, Hemichromines, and Haplochromines. Amplification was successfully observed for 29 markers (91%), with a frequency of polymorphic (P95) loci per species around 70%. The mean number of alleles per locus and species was 3.2 but varied from 3.7 within Oreochromis species to 1.6 within the nontilapia species. The high level of cross-species amplification and polymorphism of the microsatellite markers tested in this study provides powerful tools for a wide range of molecular genetic studies within tilapia species as well as for other African cichlids. PMID:22701809

  20. Amplification, Decoherence, and the Acquisition of Information by Spin Environments

    NASA Astrophysics Data System (ADS)

    Zwolak, Michael; Riedel, C. Jess; Zurek, Wojciech H.

    2016-05-01

    Quantum Darwinism recognizes the role of the environment as a communication channel: Decoherence can selectively amplify information about the pointer states of a system of interest (preventing access to complementary information about their superpositions) and can make records of this information accessible to many observers. This redundancy explains the emergence of objective, classical reality in our quantum Universe. Here, we demonstrate that the amplification of information in realistic spin environments can be quantified by the quantum Chernoff information, which characterizes the distinguishability of partial records in individual environment subsystems. We show that, except for a set of initial states of measure zero, the environment always acquires redundant information. Moreover, the Chernoff information captures the rich behavior of amplification in both finite and infinite spin environments, from quadratic growth of the redundancy to oscillatory behavior. These results will considerably simplify experimental testing of quantum Darwinism, e.g., using nitrogen vacancies in diamond.

  1. Amplification, Decoherence, and the Acquisition of Information by Spin Environments

    PubMed Central

    Zwolak, Michael; Riedel, C. Jess; Zurek, Wojciech H.

    2016-01-01

    Quantum Darwinism recognizes the role of the environment as a communication channel: Decoherence can selectively amplify information about the pointer states of a system of interest (preventing access to complementary information about their superpositions) and can make records of this information accessible to many observers. This redundancy explains the emergence of objective, classical reality in our quantum Universe. Here, we demonstrate that the amplification of information in realistic spin environments can be quantified by the quantum Chernoff information, which characterizes the distinguishability of partial records in individual environment subsystems. We show that, except for a set of initial states of measure zero, the environment always acquires redundant information. Moreover, the Chernoff information captures the rich behavior of amplification in both finite and infinite spin environments, from quadratic growth of the redundancy to oscillatory behavior. These results will considerably simplify experimental testing of quantum Darwinism, e.g., using nitrogen vacancies in diamond. PMID:27193389

  2. Heavy metal release from different ashes during serial batch tests using water and acid.

    PubMed

    Ludwig, Bernard; Khanna, Partap; Prenzel, Jürgen; Beese, Friedrich

    2005-01-01

    Most ashes contain a significant amount of heavy metals and when released from disposed or used ash materials, they can form a major environmental concern for underground waters. The use of water extracts to assess the easily mobilisable content of heavy metals may not provide an appropriate measure. This study describes the patterns of heavy metal release from ash materials in context with results from the German standard extraction method DIN-S4 (DIN 38 414 S4). Samples of four different ashes (municipal solid waste incineration ash, wood ash, brown coal ash and hard coal ash) were subjected to a number of serial batch tests with liquid renewal, some of which involved the addition of acid to neutralize carbonates and oxides. Release of heavy metals showed different patterns depending on the element, the type of material, the method of extraction and the type of the extractant used. Only a small fraction of the total heavy metal contents occurred as water soluble salts; of special significance was the amount of Cr released from the wood ash. The reaction time (1, 24 or 72 h between each extraction step with water) had only a small effect on the release of heavy metals. However, the release of most of the heavy metals was governed by the dissolution processes following proton inputs, indicating that pH-dependent tests such as CEN TC 292 or others are required to estimate long-term effects of heavy metal releases from ashes. Based on the chemical characteristics of ash materials in terms of their form and solubility of heavy metals, recommendations were made on the disposal or use of the four ash materials.

  3. Predicting anticancer peptides with Chou's pseudo amino acid composition and investigating their mutagenicity via Ames test.

    PubMed

    Hajisharifi, Zohre; Piryaiee, Moien; Mohammad Beigi, Majid; Behbahani, Mandana; Mohabatkar, Hassan

    2014-01-21

    Cancer is an important reason of death worldwide. Traditional cytotoxic therapies, such as radiation and chemotherapy, are expensive and cause severe side effects. Currently, design of anticancer peptides is a more effective way for cancer treatment. So there is a need to develop a computational method for predicting the anticancer peptides. In the present study, two methods have been developed to predict these peptides using support vector machine (SVM) as a powerful machine learning algorithm. Classifiers have been applied based on the concept of Chou's pseudo-amino acid composition (PseAAC) and local alignment kernel. Since a number of HIV-1 proteins have cytotoxic effect, therefore we predicted the anticancer effect of HIV-1 p24 protein with these methods. After the prediction, mutagenicity of 2 anticancer peptides and 2 non-anticancer peptides was investigated by Ames test. Our results show that, the accuracy and the specificity of local alignment kernel based method are 89.7% and 92.68%, respectively. The accuracy and specificity of PseAAC-based method are 83.82% and 85.36%, respectively. By computational analysis, out of 22 peptides of p24 protein, 4 peptides are anticancer and 18 are non-anticancer. In the Ames test results, it is clear that anticancer peptides (ARP788.8 and ARP788.21) are not mutagenic. Therefore the results demonstrate that the described computation methods are useful to identify potential anticancer peptides, which are worthy of further experimental validation and 2 peptides (ARP788.8 and ARP788.21) of HIV-1 p24 protein can be used as new anticancer candidates without mutagenicity.

  4. External quality assurance of malaria nucleic acid testing for clinical trials and eradication surveillance.

    PubMed

    Murphy, Sean C; Hermsen, Cornelus C; Douglas, Alexander D; Edwards, Nick J; Petersen, Ines; Fahle, Gary A; Adams, Matthew; Berry, Andrea A; Billman, Zachary P; Gilbert, Sarah C; Laurens, Matthew B; Leroy, Odile; Lyke, Kristen E; Plowe, Christopher V; Seilie, Annette M; Strauss, Kathleen A; Teelen, Karina; Hill, Adrian V S; Sauerwein, Robert W

    2014-01-01

    Nucleic acid testing (NAT) for malaria parasites is an increasingly recommended diagnostic endpoint in clinical trials of vaccine and drug candidates and is also important in surveillance of malaria control and elimination efforts. A variety of reported NAT assays have been described, yet no formal external quality assurance (EQA) program provides validation for the assays in use. Here, we report results of an EQA exercise for malaria NAT assays. Among five centers conducting controlled human malaria infection trials, all centers achieved 100% specificity and demonstrated limits of detection consistent with each laboratory's pre-stated expectations. Quantitative bias of reported results compared to expected results was generally <0.5 log10 parasites/mL except for one laboratory where the EQA effort identified likely reasons for a general quantitative shift. The within-laboratory variation for all assays was low at <10% coefficient of variation across a range of parasite densities. Based on this study, we propose to create a Molecular Malaria Quality Assessment program that fulfills the need for EQA of malaria NAT assays worldwide.

  5. Examination of the cervix with the naked eye using acetic acid test.

    PubMed

    Ottaviano, M; La Torre, P

    1982-05-15

    Examination of the cervix was carried out on 2,400 patients, by use of acetic acid test with the naked eye and the colposcope. The physiologic transformation zone was clearly identified both with the naked eye and the colposcope in 1,568 of 1,594 (99%) cases. Colposcopic examination was unsatisfactory in 108 of the 264 (41%) patients in whom the cervix was completely covered by normal squamous epithelium. An atypical transformation zone (ATZ) was identified with the naked eye as white epithelium in 98.4% and as "suspicious" in 1.6% of 312 colposcopically controlled cases. An unsatisfactory colposcopic examination occurred in 39 of the 312 (12.5%) patients with an ATZ. Final histologic diagnosis for 312 ATZs was benign lesion in 169 of 312 (54.2%), cervical intraepithelial neoplasia (CIN) grades 1 and 2 in 81 of 312 (26%), grade 3 CIN in 56 of 312 (17.9%), and preclinical invasive carcinoma in 6 of 312 (1.9%). The detection of intraepithelial or preclinical invasive cervical neoplasias should not depend on the possession of a colposcope. On the other hand, the use of a colposcope is essential for the selection of CIN that can be treated with ultraconservative therapy or with colposcopically directed conization.

  6. The Spatial Pattern of Cochlear Amplification

    PubMed Central

    Fisher, Jonathan A.N.; Nin, Fumiaki; Reichenbach, Tobias; Uthaiah, Revathy C.; Hudspeth, A.J.

    2012-01-01

    SUMMARY Sensorineural hearing loss, which stems primarily from the failure of mechanosensory hair cells, changes the traveling waves that transmit acoustic signals along the cochlea. However, the connection between cochlear mechanics and the amplificatory function of hair cells remains unclear. Using an optical technique that permits the targeted inactivation of prestin, a protein of outer hair cells that generates forces on the basilar membrane, we demonstrate that these forces interact locally with cochlear traveling waves to achieve enormous mechanical amplification. By perturbing amplification in narrow segments of the basilar membrane, we further show that a cochlear traveling wave accumulates gain as it approaches its peak. Analysis of these results indicates that cochlear amplification produces negative damping that counters the viscous drag impeding traveling waves; targeted photoinactivation locally interrupts this compensation. These results reveal the locus of amplification in cochlear traveling waves and connect the characteristics of normal hearing to molecular forces. PMID:23217746

  7. Earthquake ground motion amplification for surface waves

    NASA Astrophysics Data System (ADS)

    Bowden, Daniel C.; Tsai, Victor C.

    2017-01-01

    Surface waves from earthquakes are known to cause strong damage, especially for larger structures such as skyscrapers and bridges. However, common practice in characterizing seismic hazard at a specific site considers the effect of near-surface geology on only vertically propagating body waves. Here we show that surface waves have a unique and different frequency-dependent response to known geologic structure and that this amplification can be analytically calculated in a manner similar to current hazard practices. Applying this framework to amplification in the Los Angeles Basin, we find that peak ground accelerations for certain large regional earthquakes are underpredicted if surface waves are not properly accounted for and that the frequency of strongest ground motion amplification can be significantly different. Including surface-wave amplification in hazards calculations is therefore essential for accurate predictions of strong ground motion for future San Andreas Fault ruptures.

  8. A Simple Structure for Signal Amplification

    NASA Astrophysics Data System (ADS)

    Ding, Wan-Xiang; Gu, Chang-Gui; Liang, Xiao-Ming

    2016-02-01

    It has been found that a triple-node feed-forward motif has a function of signal amplification, where two input nodes receive the external weak signal and jointly modulate the response of the third output node [Liang et al., Phys. Rev. E 88 (2013) 012910]. We here show that the signal amplification can be further enhanced by adding a link between the two input nodes in the feed-forward motif. We further reveal that the coupling strength of the link regulates the enhancement of signal amplification in the modified feed-forward motif. We finally analyze the mechanism of signal amplification of such simple structure. Supported by the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning under Grant No. QD2015016, the National Natural Science Foundation of China under Grant Nos. 11505114 and 11305078

  9. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES - Book Chapter

    EPA Science Inventory

    This book chapter contains the following headings and subheadings: Introduction; Experimental Approach - Precautions, Template, Primers, Reaction Conditions, Enhancers, Post Amplification; Procedures - Template DNA, Basic PCR, Thermal Cycle Parameters, Enzyme Addition, Agarose Ge...

  10. Coupled isothermal polynucleotide amplification and translation system

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor)

    1998-01-01

    A cell-free system for polynucleotide amplification and translation is disclosed. Also disclosed are methods for using the system and a composition which allows the various components of the system to function under a common set of reaction conditions.

  11. The spatial pattern of cochlear amplification.

    PubMed

    Fisher, Jonathan A N; Nin, Fumiaki; Reichenbach, Tobias; Uthaiah, Revathy C; Hudspeth, A J

    2012-12-06

    Sensorineural hearing loss, which stems primarily from the failure of mechanosensory hair cells, changes the traveling waves that transmit acoustic signals along the cochlea. However, the connection between cochlear mechanics and the amplificatory function of hair cells remains unclear. Using an optical technique that permits the targeted inactivation of prestin, a protein of outer hair cells that generates forces on the basilar membrane, we demonstrate that these forces interact locally with cochlear traveling waves to achieve enormous mechanical amplification. By perturbing amplification in narrow segments of the basilar membrane, we further show that a cochlear traveling wave accumulates gain as it approaches its peak. Analysis of these results indicates that cochlear amplification produces negative damping that counters the viscous drag impeding traveling waves; targeted photoinactivation locally interrupts this compensation. These results reveal the locus of amplification in cochlear traveling waves and connect the characteristics of normal hearing to molecular forces.

  12. Cyclic Amplification of Prion Protein Misfolding

    PubMed Central

    Barria, Marcelo A; Gonzalez-Romero, Dennisse; Soto, Claudio

    2014-01-01

    Protein Misfolfing Cyclic amplification (PMCA) is a technique that take advantage of the nucleation-dependent prion replication process to accelerate the conversion of PrPC into PrPSc in the test tube. PMCA uses ultrasound waves to fragment the PrPSc polymers, increasing the amount of seeds present in the infected sample without affecting their ability to act as conversion nucleus. Over the past 5 years PMCA has became an invaluable technique to study diverse aspects of prions. The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the cellular factors involved in prion propagation, the intriguing phenomena of prion strains and species barriers, to detect PrPSc in tissues and biological fluids and to screen for inhibitors against prion replication. In this article we describe a detailed protocol of the PMCA technique, highlighting some of the important technical aspects to obtain a successful and reproducible application of the technology. PMID:22528092

  13. Rolling circle amplification of metazoan mitochondrialgenomes

    SciTech Connect

    Simison, W. Brian; Lindberg, D.R.; Boore, J.L.

    2005-07-31

    Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.

  14. Amplification of chromosomal DNA in situ

    DOEpatents

    Christian, Allen T.; Coleman, Matthew A.; Tucker, James D.

    2002-01-01

    Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

  15. Extended Gate Field-Effect Transistor Biosensors for Point-Of-Care Testing of Uric Acid.

    PubMed

    Guan, Weihua; Reed, Mark A

    2017-01-01

    An enzyme-free redox potential sensor using off-chip extended-gate field effect transistor (EGFET) with a ferrocenyl-alkanethiol modified gold electrode has been used to quantify uric acid concentration in human serum and urine. Hexacyanoferrate (II) and (III) ions are used as redox reagent. The potentiometric sensor measures the interface potential on the ferrocene immobilized gold electrode, which is modulated by the redox reaction between uric acid and hexacyanoferrate ions. The device shows a near Nernstian response to uric acid and is highly specific to uric acid in human serum and urine. The interference that comes from glucose, bilirubin, ascorbic acid, and hemoglobin is negligible in the normal concentration range of these interferents. The sensor also exhibits excellent long term reliability and is regenerative. This extended gate field effect transistor based sensor is promising for point-of-care detection of uric acid due to the small size, low cost, and low sample volume consumption.

  16. Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers.

    PubMed

    Jauset-Rubio, Miriam; Svobodová, Markéta; Mairal, Teresa; McNeil, Calum; Keegan, Neil; El-Shahawi, Mohammad S; Bashammakh, Abdulaziz S; Alyoubi, Abdulrahman O; O'Sullivan, Ciara K

    2016-11-01

    In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which β-conglutin immobilized on the test line of a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the β-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized β-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

  17. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

    PubMed

    Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

    2016-10-01

    Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR.

  18. Amplification uncertainty relation for probabilistic amplifiers

    NASA Astrophysics Data System (ADS)

    Namiki, Ryo

    2015-09-01

    Traditionally, quantum amplification limit refers to the property of inevitable noise addition on canonical variables when the field amplitude of an unknown state is linearly transformed through a quantum channel. Recent theoretical studies have determined amplification limits for cases of probabilistic quantum channels or general quantum operations by specifying a set of input states or a state ensemble. However, it remains open how much excess noise on canonical variables is unavoidable and whether there exists a fundamental trade-off relation between the canonical pair in a general amplification process. In this paper we present an uncertainty-product form of amplification limits for general quantum operations by assuming an input ensemble of Gaussian-distributed coherent states. It can be derived as a straightforward consequence of canonical uncertainty relations and retrieves basic properties of the traditional amplification limit. In addition, our amplification limit turns out to give a physical limitation on probabilistic reduction of an Einstein-Podolsky-Rosen uncertainty. In this regard, we find a condition that probabilistic amplifiers can be regarded as local filtering operations to distill entanglement. This condition establishes a clear benchmark to verify an advantage of non-Gaussian operations beyond Gaussian operations with a feasible input set of coherent states and standard homodyne measurements.

  19. Onshore seismic amplifications due to bathymetric features

    NASA Astrophysics Data System (ADS)

    Rodríguez-Castellanos, A.; Carbajal-Romero, M.; Flores-Guzmán, N.; Olivera-Villaseñor, E.; Kryvko, A.

    2016-08-01

    We perform numerical calculations for onshore seismic amplifications, taking into consideration the effect of bathymetric features on the propagation of seismic movements. To this end, the boundary element method is applied. Boundary elements are employed to irradiate waves and, consequently, force densities can be obtained for each boundary element. From this assumption, Huygens’ principle is applied, and since the diffracted waves are built at the boundary from which they are radiated, this idea is equivalent to Somigliana’s representation theorem. The application of boundary conditions leads to a linear system being obtained (Fredholm integral equations). Several numerical models are analyzed, with the first one being used to verify the proposed formulation, and the others being used to estimate onshore seismic amplifications due to the presence of bathymetric features. The results obtained show that compressional waves (P-waves) generate onshore seismic amplifications that can vary from 1.2 to 5.2 times the amplitude of the incident wave. On the other hand, the shear waves (S-waves) can cause seismic amplifications of up to 4.0 times the incident wave. Furthermore, an important result is that in most cases the highest seismic amplifications from an offshore earthquake are located on the shoreline and not offshore, despite the seafloor configuration. Moreover, the influence of the incident angle of seismic waves on the seismic amplifications is highlighted.

  20. Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology.

    PubMed

    Daher, Rana K; Stewart, Gale; Boissinot, Maurice; Boudreau, Dominique K; Bergeron, Michel G

    2015-04-01

    Recombinase polymerase amplification (RPA) technology relies on three major proteins, recombinase proteins, single-strand binding proteins, and polymerases, to specifically amplify nucleic acid sequences in an isothermal format. The performance of RPA with respect to sequence mismatches of closely-related non-target molecules is not well documented and the influence of the number and distribution of mismatches in DNA sequences on RPA amplification reaction is not well understood. We investigated the specificity of RPA by testing closely-related species bearing naturally occurring mismatches for the tuf gene sequence of Pseudomonas aeruginosa and/or Mycobacterium tuberculosis and for the cfb gene sequence of Streptococcus agalactiae. In addition, the impact of the number and distribution of mismatches on RPA efficiency was assessed by synthetically generating 14 types of mismatched forward primers for detecting five bacterial species of high diagnostic relevance such as Clostridium difficile, Staphylococcus aureus, S. agalactiae, P. aeruginosa, and M. tuberculosis as well as Bacillus atropheus subsp. globigii for which we use the spores as internal control in diagnostic assays. A total of 87 mismatched primers were tested in this study. We observed that target specific RPA primers with mismatches (n > 1) at their 3'extrimity hampered RPA reaction. In addition, 3 mismatches covering both extremities and the center of the primer sequence negatively affected RPA yield. We demonstrated that the specificity of RPA was multifactorial. Therefore its application in clinical settings must be selected and validated a priori. We recommend that the selection of a target gene must consider the presence of closely-related non-target genes. It is advisable to choose target regions with a high number of mismatches (≥36%, relative to the size of amplicon) with respect to closely-related species and the best case scenario would be by choosing a unique target gene.