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Sample records for acid amplification method

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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).

  9. 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).

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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).

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. [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.

  3. 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…

  4. 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.

  5. 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.

  6. 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.

  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. [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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. Method Of Signal Amplification In Multi-Chromophore Luminescence Sensors

    DOEpatents

    Levitsky, Igor A.; Krivoshlykov, Sergei G.

    2004-02-03

    A fluorescence-based method for highly sensitive and selective detection of analyte molecules is proposed. The method employs the energy transfer between two or more fluorescent chromophores in a carefully selected polymer matrix. In one preferred embodiment, signal amplification has been achieved in the fluorescent sensing of dimethyl methylphosphonate (DMMP) using two dyes, 3-aminofluoranthene (AM) and Nile Red (NR), in a hydrogen bond acidic polymer matrix. The selected polymer matrix quenches the fluorescence of both dyes and shifts dye emission and absorption spectra relative to more inert matrices. Upon DMMP sorption, the AM fluorescence shifts to the red at the same time the NR absorption shifts to the blue, resulting in better band overlap and increased energy transfer between chromophores. In another preferred embodiment, the sensitive material is incorporated into an optical fiber system enabling efficient excitation of the dye and collecting the fluorescent signal form the sensitive material on the remote end of the system. The proposed method can be applied to multichromophore luminescence sensor systems incorporating N-chromophores leading to N-fold signal amplification and improved selectivity. The method can be used in all applications where highly sensitive detection of basic gases, such as dimethyl methylphosphonate (DMMP), Sarin, Soman and other chemical warfare agents having basic properties, is required, including environmental monitoring, chemical industry and medicine.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Alternative Chemical Amplification Methods for Peroxy Radical Detection

    NASA Astrophysics Data System (ADS)

    Wood, E. C. D.

    2014-12-01

    Peroxy radicals (HO2, CH3O2, etc.) are commonly detected by the chemical amplification technique, in which ambient air is mixed with high concentrations of CO and NO, initiating a chain reaction that produces 30 - 200 NO2 molecules per sampled peroxy radical. The NO2 is then measured by one of several techniques. With the exception of CIMS-based techniques, the chemical amplification method has undergone only incremental improvements since it was first introduced in 1982. The disadvantages of the technique include the need to use high concentrations of CO and the greatly reduced sensitivity of the amplification chain length in the presence of water vapor. We present a new chemical amplification scheme in which either ethane or acetaldehyde is used in place of CO, with the NO2 product detected using Cavity Attenuated Phase Shift spectroscopy (CAPS). Under dry conditions, the amplification factor of the alternative amplifiers are approximately six times lower than the CO-based amplifier. The relative humidity "penalty" is not as severe, however, such that at typical ambient relative humidity (RH) values the amplification factor is within a factor of three of the CO-based amplifier. Combined with the NO2 sensitivity of CAPS and a dual-channel design, the detection limit of the ethane amplifier is less than 2 ppt (1 minute average, signal-to-noise ratio 2). The advantages of these alternative chemical amplification schemes are improved safety, a reduced RH correction, and increased sensitivity to organic peroxy radicals relative to HO2.

  3. Nucleic acid arrays and methods of synthesis

    DOEpatents

    Sabanayagam, Chandran R.; Sano, Takeshi; Misasi, John; Hatch, Anson; Cantor, Charles

    2001-01-01

    The present invention generally relates to high density nucleic acid arrays and methods of synthesizing nucleic acid sequences on a solid surface. Specifically, the present invention contemplates the use of stabilized nucleic acid primer sequences immobilized on solid surfaces, and circular nucleic acid sequence templates combined with the use of isothermal rolling circle amplification to thereby increase nucleic acid sequence concentrations in a sample or on an array of nucleic acid sequences.

  4. 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.

  5. 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

  6. Advances in nucleic acid-based detection methods.

    PubMed Central

    Wolcott, M J

    1992-01-01

    Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in clinical settings. After the potential of polymerase chain reaction became apparent, other methods of nucleic acid amplification and detection were developed. These alternative nucleic acid amplification methods may become serious contenders for application to routine laboratory analyses. This review presents some background information on nucleic acid analyses that might be used in clinical and anatomical laboratories and describes some recent advances in the amplification and detection of nucleic acids. PMID:1423216

  7. Study Unveils New Method for Universal Extraction and PCR Amplification of Fungal DNA

    DTIC Science & Technology

    2014-06-12

    rare or hard to identify fungal infections. The new extraction and amplification method can be universally applied to fungi , according to the...best treatments. In addition, rare fungi , or species with phenotypic doppelgangers, can stump medical mycologists, so molecular methods are critical...However, isolating DNA from fungi can be problematic, and an inexpensive method to isolate and amplify nucleic acids from all types of pathogenic fungi

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. A new ultrasonic signal amplification method for detection of bacteria

    NASA Astrophysics Data System (ADS)

    Kant Shukla, Shiva; Resa López, Pablo; Sierra Sánchez, Carlos; Urréjola, José; Segura, Luis Elvira

    2012-10-01

    A new method is presented that increases the sensitivity of ultrasound-based techniques for detection of bacteria. The technique was developed for the detection of catalase-positive microorganisms. It uses a bubble trapping medium containing hydrogen peroxide that is mixed with the sample for microbiological evaluation. The enzyme catalase is present in catalase-positive bacteria, which induces a rapid hydrolysis of hydrogen peroxide, forming bubbles which remain in the medium. This reaction results in the amplification of the mechanical changes that the microorganisms produce in the medium. The effect can be detected by means of ultrasonic wave amplitude continuous measurement since the bubbles increase the ultrasonic attenuation significantly. It is shown that microorganism concentrations of the order of 105 cells ml-1 can be detected using this method. This allows an improvement of three orders of magnitude in the ultrasonic detection threshold of microorganisms in conventional culture media, and is competitive with modern rapid microbiological methods. It can also be used for the characterization of the enzymatic activity.

  1. 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.

  2. 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.

  3. 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.

  4. Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

    PubMed

    Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark

    2016-06-01

    Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.

  5. 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.

  6. Amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded DNA viruses.

    PubMed

    Kim, Kyoung-Ho; Bae, Jin-Woo

    2011-11-01

    Investigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes. In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral diversity.

  7. 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.

  8. 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

  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. Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an in...

  11. Models and methods to characterize site amplification from a pair of records

    USGS Publications Warehouse

    Safak, E.

    1997-01-01

    The paper presents a tutorial review of the models and methods that are used to characterize site amplification from the pairs of rock- and soil-site records, and introduces some new techniques with better theoretical foundations. The models and methods discussed include spectral and cross-spectral ratios, spectral ratios for downhole records, response spectral ratios, constant amplification factors, parametric models, physical models, and time-varying filters. An extensive analytical and numerical error analysis of spectral and cross-spectral ratios shows that probabilistically cross-spectral ratios give more reliable estimates of site amplification. Spectral ratios should not be used to determine site amplification from downhole-surface recording pairs because of the feedback in the downhole sensor. Response spectral ratios are appropriate for low frequencies, but overestimate the amplification at high frequencies. The best method to be used depends on how much precision is required in the estimates.

  12. 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.

  13. 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

  14. 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.

  15. The uses and limitations of the square‐root‐impedance method for computing site amplification

    USGS Publications Warehouse

    Boore, David

    2013-01-01

    The square‐root‐impedance (SRI) method is a fast way of computing approximate site amplification that does not depend on the details from velocity models. The SRI method underestimates the peak response of models with large impedance contrasts near their base, but the amplifications for those models is often close to or equal to the root mean square of the theoretical full resonant (FR) response of the higher modes. On the other hand, for velocity models made up of gradients, with no significant impedance changes across small ranges of depth, the SRI method systematically underestimates the theoretical FR response over a wide frequency range. For commonly used gradient models for generic rock sites, the SRI method underestimates the FR response by about 20%–30%. Notwithstanding the persistent underestimation of amplifications from theoretical FR calculations, however, amplifications from the SRI method may often provide more useful estimates of amplifications than the FR method, because the SRI amplifications are not sensitive to details of the models and will not exhibit the many peaks and valleys characteristic of theoretical full resonant amplifications (jaggedness sometimes not seen in amplifications based on averages of site response from multiple recordings at a given site). The lack of sensitivity to details of the velocity models also makes the SRI method useful in comparing the response of various velocity models, in spite of any systematic underestimation of the response. The quarter‐wavelength average velocity, which is fundamental to the SRI method, is useful by itself in site characterization, and as such, is the fundamental parameter used to characterize the site response in a number of recent ground‐motion prediction equations.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. A Novel Real-Time DNA Detection System for Loop-Mediated Isothermal Amplification Method

    NASA Astrophysics Data System (ADS)

    Kakugawa, Koji; Yamada, Kenji; Maeda, Hiroshi; Takashiba, Shougo

    We developed a novel real-time DNA detection system for loop-mediated isothermal amplification (LAMP) method. Our prototype was composed of a thermostatic chamber, a hole slide glass, LED and a web camera. The reaction mixture was injected into the slide glass hole and the LAMP reaction was carried out at 63°C for 2 hours. To observe the DNA amplification, we monitored the fluorescence intensity of SYBR Green I that was excited by the blue LED. The captured BMP images were analyzed by NIH Image J software. The DNA amplification and amplification monitoring experiment was successful. Furthermore, quantitative accuracy was evaluated based on real-time PCR. The reaction time correlates well with the DNA concentration. These results indicate the successful development of a novel real-time DNA detection system for LAMP method.

  1. Methods and devices based on brillouin selective sideband amplification

    NASA Technical Reports Server (NTRS)

    Yao, X. Steve (Inventor)

    2003-01-01

    Opto-electronic devices and techniques using Brillouin scattering to select a sideband in a modulated optical carrier signal for amplification. Two lasers respectively provide a carrier signal beam and a Brillouin pump beam which are fed into an Brillouin optical medium in opposite directions. The relative frequency separation between the lasers is adjusted to align the frequency of the backscattered Brillouin signal with a desired sideband in the carrier signal to effect a Brillouin gain on the sideband. This effect can be used to implement photonic RF signal mixing and conversion with gain, conversion from phase modulation to amplitude modulation, photonic RF frequency multiplication, optical and RF pulse generation and manipulation, and frequency-locking of lasers.

  2. A method for selective PCR-amplification of genomic DNA fragments (SAGF method)

    SciTech Connect

    Zheleznaya, L.A.; Menzenyuk, O.Y.; Matvienko, N.N.; Matvienko, N.I.

    1995-09-01

    A method is suggested for dividing into individual sets of the complex mixtures of fragments obtained by DNA cleavage with type IIS and IIN restriction endonucleases producing single-stranded termini with different sequences at the DNA fragment ends. The method is based on the ligation of short double-stranded adapters with single-stranded ends complementary to termini of the selected set of fragments followed by PCR-amplification with the primer representing one of the adapter chains. Using endonucleases BcoKI and Bli736I, recognizing sequences CTCTTC and GGTCTC and producing three- and four nucleotide 5{prime}-termini, respectively, it has been shown that amplification of a set of fragments occurs only upon attachment of the adapters to the DNA fragments with DNA-ligase. Several possible applications of the SAGF method are suggested: obtaining individual bands in DNA fingerprinting; reducing the kinetic complexity of DNA in representative difference analysis (RDA method) of complex genomes; cataloging of DNA fragments; construction of physical genome maps. 13 refs., 3 figs., 2 tabs.

  3. Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus.

    PubMed

    Tsai, Shinn Shyong; Chang, Yeng Ling; Huang, Yen Li; Liu, Hung Jen; Ke, Guan Ming; Chiou, Chwei Jang; Hsieh, Yao Ching; Chang, Tsung Chou; Cheng, Li Ting; Chuang, Kuo Pin

    2014-05-01

    There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity.

  4. 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.

  5. 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.

  6. 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).

  7. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

    PubMed

    Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

    2014-12-01

    Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. Different whole-genome amplification methods as a preamplification tool in Y-chromosome Loci analysis.

    PubMed

    Maciejewska, Agnieszka; Jakubowska, Joanna; Pawłowski, Ryszard

    2014-06-01

    Degraded and low template DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded and low template DNA is of great importance, because its results impact the quality and reliability of expert testimonies. Recently, a number of whole-genome amplification (WGA) methods have been proposed as preamplification tools improving quantity and quality of DNA. We chose, investigated, and compared 7 WGA methods to evaluate their ability to "recover" degraded and nondegraded DNA. These methods include degenerate oligonucleotide primed polymerase chain reaction, primer extension preamplification (PEP) polymerase chain reaction, GenomePlex WGA (Sigma), multiple displacement amplification, GenomiPhi Amplification Kit (Amersham Biosciences), restriction and circularization aided rolling circle amplification, and blunt-end ligation-mediated WGA. Recently, we have described the comparison of these methods' efficiency and reliability using SGMPlus kit. However, Y-chromosome profiling is also often used in analysis of both nondegraded and degraded DNA. This includes criminal cases and investigation of kinship in male linage. Here we demonstrate the impact of WGA methods on Y-chromosome loci (Yfiler) reactivation.The best results for nondegraded DNA were obtained with GenomiPhi kit and PEP method. In the case of degraded DNA (200 base pairs), the most complete profiles were obtained with GenomePlex kit and PEP method. None of the analyzed methods allowed full reactivation of degraded (200 base pairs) DNA in terms of Y-chromosome loci profiling.

  13. Computational method and system for modeling, analyzing, and optimizing DNA amplification and synthesis

    DOEpatents

    Vandersall, Jennifer A.; Gardner, Shea N.; Clague, David S.

    2010-05-04

    A computational method and computer-based system of modeling DNA synthesis for the design and interpretation of PCR amplification, parallel DNA synthesis, and microarray chip analysis. The method and system include modules that address the bioinformatics, kinetics, and thermodynamics of DNA amplification and synthesis. Specifically, the steps of DNA selection, as well as the kinetics and thermodynamics of DNA hybridization and extensions, are addressed, which enable the optimization of the processing and the prediction of the products as a function of DNA sequence, mixing protocol, time, temperature and concentration of species.

  14. Method for isolating nucleic acids

    SciTech Connect

    Hurt, Jr., Richard Ashley; Elias, Dwayne A.

    2015-09-29

    The current disclosure provides methods and kits for isolating nucleic acid from an environmental sample. The current methods and compositions further provide methods for isolating nucleic acids by reducing adsorption of nucleic acids by charged ions and particles within an environmental sample. The methods of the current disclosure provide methods for isolating nucleic acids by releasing adsorbed nucleic acids from charged particles during the nucleic acid isolation process. The current disclosure facilitates the isolation of nucleic acids of sufficient quality and quantity to enable one of ordinary skill in the art to utilize or analyze the isolated nucleic acids for a wide variety of applications including, sequencing or species population analysis.

  15. 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

  16. [Development of uncompetitive exogenous internal amplification control for real-time PCR based on UFA method].

    PubMed

    Ivanov, M K; Bragin, A G; Prasolova, M A; Vedernikov, V E; Dymshits, G M

    2009-01-01

    An uncompetitive exogenous internal amplification control method (EIAC) was developed on the basis of short synthetic DNA segment, whose amplification can be detected in real time by UFA spectroscopy principle. The EIAC was shown to be useful as internal control in diagnostic test systems based on DNA or RNA detection by multiplex real-time PCR. It can be applied to assess the quality of extracted DNA or RNA, and also to detect and study the factors causing PCR inhibition and earlier plateau effect.

  17. 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.

  18. Nucleic Acid Detection Methods

    DOEpatents

    Smith, Cassandra L.; Yaar, Ron; Szafranski, Przemyslaw; Cantor, Charles R.

    1998-05-19

    The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3'-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated.

  19. Nucleic acid detection methods

    DOEpatents

    Smith, C.L.; Yaar, R.; Szafranski, P.; Cantor, C.R.

    1998-05-19

    The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3{prime}-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated. 18 figs.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. Chemical amplification methods for the sequential determination of trace amounts of ruthenium by titrimetric and spectrophotometric procedures.

    PubMed

    El-Shahawi, M S; Barakat, S A

    1995-11-01

    Two simple, inexpensive and rapid iodometric and spectrophotometric procedures were developed for trace amount determination of ruthenium. The proposed methods were based on the oxidation of ruthenium(II or III) with sodium periodate at pH 2.4-3.6, masking the excess periodate with sodium molybdate. The released iodate was then allowed to react with KI at pH 3, with subsequent determination of the released iodine spectrophotometry as triiodide at 350 nm or iodometry with 0.005 M sodium thiosulphate. This procedure offers an 18- and 15-fold amplification per Ru(II) or Ru(III) ion, respectively. Alternatively, the produced iodine was extracted with CHCl(3), shaken with an aqueous solution of sodium sulphite and the produced iodide ion was then allowed to react with bromine (or sodium periodate). The released iodate was subsequently determined by iodometry or spectrophotometry after addition of KI. The bromine and sodium periodate oxidation procedures offered 90- and 360-fold amplification per ruthenium(III) ion, and 108- and 432-fold amplification per ruthenium(II) ion. Ruthenium(IV) content was determined by these procedures after prior reduction to Ru(III) with sulphurous acid. The binary mixtures Ru(II)-Ru(III); Ru(III)-Ru(IV) and Ru(II)-Ru(IV) in aqueous solution at concentration 0.05 mug ml(-1) were successfully analyzed by the developed procedures. The utility of the proposed methods for the analysis of ruthenium in its complexes was demonstrated. Natural seawater and seawater spiked with ruthenium were analyzed satisfactorily.

  8. 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.

  9. A new PCR method: one primer amplification of PCR-CTPP products.

    PubMed

    Yin, Guang; Mitsuda, Yoko; Ezaki, Takayuki; Hamajima, Nobuyuki

    2012-10-01

    Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.

  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. 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. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

    PubMed

    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.

  13. Sensitive Method for Biomolecule Detection Utilizing Signal Amplification with Porphyrin Nanoparticles.

    PubMed

    Gibson, Lauren E; Wright, David W

    2016-06-07

    Disease diagnosis requires identification of biomarkers that occur in small quantities, making detection a difficult task. Effective diagnosis is an even greater challenge in low-resource areas of the world. Methods must be simple, stable, and sensitive so that tests can be easily administered and withstand uncontrolled environmental conditions. One approach to this issue is development of stable signal amplification strategies. In this work, we applied the nanocrystal-based signal amplification method to tetra(4-carboxyphenyl)porphyrin nanoparticles (TCPP NPs). The dissolution of the nanoparticle into thousands of porphyrin molecules results in amplified detection of the biomarker. By using nanoparticles as the signal-generating moiety, stability of the detection method is increased relative to commonly used enzyme-based assays. Additionally, the inherent fluorescent signal of TCPP molecules can be measured after nanoparticle dissolution. The ability to directly read the TCPP fluorescent signal increases assay simplicity by reducing the steps required for the test. This detection method was optimized by detecting rabbit IgG and then was applied to the detection of the malarial biomarker Plasmodium falciparum histidine-rich protein II (pfHRPII) from a complex matrix. The results for both biomarkers were assays with low picomolar limits of detection.

  14. 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.

  15. 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.

  16. 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.

  17. Highly-sensitive electrochemical immunosensing method based on dual amplification systems.

    PubMed

    Yasukawa, Tomoyuki; Yoshimoto, Yoshimi; Goto, Takuya; Mizutani, Fumio

    2012-01-01

    In this work, a novel immunosensing method has been developed on the basis of the sensitive determination of a product generated by an enzyme reaction with dual amplification system combining an electrochemical-redox cycling and coulometric signal transduction using a galvanic cell. Analytes were captured on microparticles to form sandwich-type immunocomplexes and then labeled with β-galactosidase (β-gal). 4-Aminophenol (PAP) produced by enzyme reaction of β-gal was introduced into the anode compartment consisting of a comb type of an interdigitated array (IDA) electrode. PAP was oxidized at the IDA electrode by the coupled reduction of silver ions at the glassy carbon (GC) electrode of the cathode, resulting in the deposition of silver metal on the GC electrode. The other comb of the IDA electrode was used to reduce quinoneimine generated by the oxidation of PAP, regenerating PAP. The deposited silver was collectively converted to a signal by anodic stripping voltammetry. The amount of silver deposited corresponded to the degree of PAP oxidation by redox cycling, which leads to an enhancement of the stripping signal due to the conversion of the product (PAP) and accumulation of the insoluble silver metal. Using carcinoembryonic antigen as a model analyte, the present immunosensing method showed linear behavior over two orders of magnitude with detection limits down to 0.01 ng/mL. Dual signal amplification with redox cycling and coulometric signal transduction provides a promising, sensitive, and simple method for the determination of marker proteins.

  18. A loop-mediated isothermal amplification method for rapid detection of NDM-1 gene.

    PubMed

    Qi, Jing; Du, Yijun; Zhu, Xiaoling; Bai, Hua; Luo, Yanbo; Liu, Yuqing

    2012-08-01

    A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40 min under isothermal conditions at 65°C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1 pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Development of a rapid cyprinid herpesvirus 2 detection method by loop-mediated isothermal amplification.

    PubMed

    Liang, L-G; Xie, J; Luo, D

    2014-10-01

    Cyprinid herpesvirus 2 (CyHV2) is a pathogen that causes severe disease and high mortality in goldfish and Prussian carp. We developed a six primer loop-mediated isothermal amplification (LAMP) assay targeting the intercapsomeric triplex protein gene. CyHV-2 DNA was 10-fold serially diluted (10(8)-10(0) copies μl(-1)) and was used as the template to determine primer sensitivity. LAMP assays were performed with DNA templates from other pathogens to determine specificity. The LAMP assay had an unequivocal detection limit of 10 copies μl(-1), which was 100 times lower than that of the polymerase chain reaction. Other pathogen strains were not amplified by the LAMP primers, indicating good specificity. SYBR Green I was added to visually detect the amplification products. Assay applicability was evaluated in 120 samples of Carassius auratus gibelio, and a positive rate of 92·5% was obtained. In conclusion, a conventional LAMP assay has high convenience, rapidity, sensitivity and specificity for detecting CyHV-2 in infected aquatic organisms. Significance and impact of the study: Herpesviral haematopoietic necrosis, caused by cyprinid herpesvirus 2 (CyHV-2), is a severe disease of goldfish and Prussian carp associated with high mortality. We developed a loop-mediated isothermal amplification (LAMP) assay to detect CyHV-2 at relatively low plasmid DNA copy levels. The results show that the LAMP assay has a number of advantages (simple, sensitive, rapid and specific) over the conventional polymerase chain reaction and can be applied in the laboratory and field. Particularly, the method is highly applicable to facilitate surveillance and early diagnosis of CyHV-2.

  4. Development of a loop-mediated isothermal amplification method for detecting virulent Rhodococcus equi.

    PubMed

    Kinoshita, Yuta; Niwa, Hidekazu; Higuchi, Tohru; Katayama, Yoshinari

    2016-09-01

    Rhodococcus equi is the most important causative bacterium of severe pneumonia in foals. We report herein the development of a specific loop-mediated isothermal amplification (LAMP) assay, which targets a gene encoding vapA for detecting virulent R. equi The detection limit of the LAMP assay was 10(4) colony forming units (CFU)/mL, which was equal to 10 CFU/reaction. The clinical efficacy of the LAMP assay was compared with those of 2 published PCR-based methods: nested PCR and quantitative real-time (q)PCR. Agreements between bacterial culture, which is the gold standard for detection of R. equi, and each of the 3 molecular tests were measured by calculating a kappa coefficient. The kappa coefficients of the LAMP (0.760), nested PCR (0.583), and qPCR (0.888) indicated substantial agreement, moderate agreement, and almost perfect agreement, respectively. Although the clinical efficacy of LAMP was not the best among the 3 methods tested, LAMP could be more easily introduced into less well-equipped clinics because it does not require special equipment (such as a thermocycler) for gene amplification. Veterinary practitioners could diagnose R. equi pneumonia more quickly by using LAMP and could use the results to select an appropriate initial treatment.

  5. Detection of peanut (Arachis hypogaea) allergen by Real-time PCR method with internal amplification control.

    PubMed

    Zhang, Wen-Ju; Cai, Qin; Guan, Xiao; Chen, Qin

    2015-05-01

    Specific primer sets were designed based on the DNA sequence of Ara h 1, one of the major peanut (Arachis hypogaea) allergens, and a competitive internal amplification control (IAC) was designed by compound primer technology. By choosing 314 copies/PCR as the IAC dosage, a Real-time PCR method with IAC was established for detecting peanut allergen Ara h 1 DNA. The method showed high specificity with a detection limit of 0.005% peanut. A series of commercial food products with/without peanut components were tested. Among these products, the peanut allergen Ara h 1 DNA could be detected in 12 products labelled containing peanut ingredients, in two without a declaration of peanut and one labelled that was produced in a facility that produced peanut-containing foods. This indicates that the method is highly sensitive for the detection of peanut ingredients in foods.

  6. Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP) method.

    PubMed

    Abdullah, J; Saffie, N; Sjasri, F A R; Husin, A; Abdul-Rahman, Z; Ismail, A; Aziah, I; Mohamed, M

    2014-01-01

    An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.

  7. NLA-QAM - A method for generating high-power QAM signals through nonlinear amplification

    NASA Astrophysics Data System (ADS)

    Morais, D. H.; Feher, K.

    1982-03-01

    A generalized technique for generating quadrature amplitude modulated (QAM) signals that permits nonlinear amplification is presented. With this technique a high-power (2 to the 2n power)-state QAM signal is generated by combining n unfiltered, nonlinear amplified, QPSK signals, n being a positive integer. The specific methods of generating 16- and 64-state signals using this technique are presented. An attractive feature of the technique is that despite significant difference with the conventional method of generating QAM signals, the same straightforward demodulation techniques apply to both, and the Pe versus S/N performances are essentially identical. In the 16-state version employing traveling wave tubes (TWT's) or GaAs FET amplifiers, the technique is shown to result in a transmitter output power advantage that is on the order of 5 dB compared to the conventional method. This advantage is achieved, however, at the expense of an additional output power amplifier.

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

    PubMed Central

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

    1994-01-01

    Two criteria must be met before mycobacterial specimens can be tested by DNA amplification methods: (i) the sample must be rendered noninfectious, and (ii) the organisms must be lysed to free the DNA. Previous publications reporting DNA amplification of mycobacteria have concentrated on lysis and amplification procedures and have not addressed the issue of sample safety. We have shown that heating of samples below 100 degrees C may not consistently kill mycobacteria; however, heating at 100 degrees C in a boiling-water bath or a forced-air oven for a minimum of 5 min kills mycobacteria, including Mycobacterium thermoresistibile. Furthermore, heating at 100 degrees C for 30 min consistently lyses mycobacteria to produce short fragments of DNA that are suitable for amplification by PCR and strand displacement amplification. This procedure works with clinical samples digested by the n-acetyl cysteine-NaOH method as well as with suspensions of organisms in phosphate buffer. This paper also demonstrates the feasibility of using strand displacement amplification with clinical specimens. Images PMID:7814537

  9. 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.

  10. Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

    PubMed

    Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

    2013-11-01

    The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg(-1) GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25.

  11. Loop-mediated isothermal amplification (LAMP)-based method for rapid mushroom species identification.

    PubMed

    Vaagt, Franziska; Haase, Ilka; Fischer, Markus

    2013-02-27

    Toxic mushroom species, such as the death cap ( Amanita phalloides ), are responsible for most mushroom poisonings. In the present work, novel loop-mediated isothermal amplification (LAMP) assays were used for the differentiation of even closely related edible and toxic mushroom species. The applicability of these methods was tested by cross-reaction studies and analysis of spiked mushroom samples (raw and fried material). Contaminations at the level of 2% (w/w) could be detected in different mushroom blends. Three detection methods were used: agarose gel analysis, fluorimetric real-time detection, and visual detection by lateral flow dipsticks (LFD). The LAMP assay combined with LFD detection allows the identification of A. phalloides in about 2 h (including DNA extraction) at a very low level of technical equipment (micropestle, water bath, and mobile centrifuge), which makes this technique perfectly suited for on-site applications.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. Combination of an unbiased amplification method and a resequencing microarray for detecting and genotyping equine arteritis virus.

    PubMed

    Hans, Aymeric; Gaudaire, Delphine; Manuguerra, Jean-Claude; Leon, Albertine; Gessain, Antoine; Laugier, Claire; Berthet, Nicolas; Zientara, Stephan

    2015-01-01

    This study shows that an unbiased amplification method applied to equine arteritis virus RNA significantly improves the sensitivity of the real-time reverse transcription-quantitative PCR (RT-qPCR) recommended by the World Organization for Animal Health. Twelve viral RNAs amplified using this method were hybridized on a high-density resequencing microarray for effective viral characterization.

  17. Comparison of DNA extraction methods for polymerase chain reaction amplification of guanaco (Lama guanicoe) fecal DNA samples.

    PubMed

    Espinosa, M I; Bertin, A; Squeo, F A; Cortés, A; Gouin, N

    2015-01-23

    Feces-based population genetic studies have become increasingly popular. However, polymerase chain reaction (PCR) amplification rates from fecal material vary depending on the species, populations, loci, and extraction protocols. Here, we assessed the PCR amplification success of three microsatellite markers and a segment of the mitochondrial control region of DNA extracted from field-collected feces of guanaco (Lama guanicoe) using two protocols - Qiagen DNA Stool Kit and 2 cetyltrimethylammonium bromide/phenol:chloroform:isoamyl alcohol (2CTAB/PCI) method. Chelex resin treatment to remove inhibitors was also tested. Our results show that the mitochondrial locus was the most difficult to amplify. PCR success rates improved for all markers after Chelex treatment of extracted DNA, and 2CTAB/PCI method (95.83%) appeared to perform slightly better than stool kit (91.67%) for the nuclear markers. Amplification success was significantly influenced by the extraction method, Chelex treatment, and locus (P < 0.001) but not by the freshness of the feces (fresh vs old, P = 0.17). The repeatability levels were high without Chelex treatment (> 0.89), but they decreased slightly after treatment for amplification of nuclear markers and markedly after treatment for amplification of the mitochondrial control region. Thus, we showed that Chelex treatment gives high PCR success, especially for nuclear markers, and adequate DNA extraction rates can be achieved from L. guanicoe feces even from non-fresh fecal material. Although not significant, 2CTAB/PCI method tended to provide higher successful amplification rates on a whole set of samples, suggesting that the method could be particularly useful when using small sample sizes.

  18. Single-primer-limited amplification: a method to generate random single-stranded DNA sub-library for aptamer selection.

    PubMed

    He, Chao-Zhu; Zhang, Kun-He; Wang, Ting; Wan, Qin-Si; Hu, Piao-Ping; Hu, Mei-Di; Huang, De-Qiang; Lv, Nong-Hua

    2013-09-01

    The amplification of a random single-stranded DNA (ssDNA) library by polymerase chain reaction (PCR) is a key step in each round of aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX), but it can be impeded by the amplification of by-products due to the severely nonspecific hybridizations among various sequences in the PCR system. To amplify a random ssDNA library free from by-products, we developed a novel method termed single-primer-limited amplification (SPLA), which was initiated from the amplification of minus-stranded DNA (msDNA) of an ssDNA library with reverse primer limited to 5-fold molar quantity of the template, followed by the amplification of plus-stranded DNA (psDNA) of the msDNA with forward primer limited to 10-fold molar quantity of the template and recovery of psDNA by gel excision. We found that the amount of by-products increased with the increase of template amount and thermal cycle number. With the optimized template amount and thermal cycle, SPLA could amplify target ssDNA without detectable by-products and nonspecific products and could produce psDNA 16.1 times as much as that by asymmetric PCR. In conclusion, SPLA is a simple and feasible method to efficiently generate a random ssDNA sub-library for aptamer selection.

  19. A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

    PubMed Central

    Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

    2015-01-01

    Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation

  20. Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

    PubMed Central

    Liu, Wei; Huang, Simo; Liu, Ningwei; Dong, Derong; Yang, Zhan; Tang, Yue; Ma, Wen; He, Xiaoming; Ao, Da; Xu, Yaqing; Zou, Dayang; Huang, Liuyu

    2017-01-01

    This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25–45 bp, beacon concentration of 0.6–1 pmol/μL, and reaction temperature of 60–65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product. PMID:28059137

  1. 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.

  2. 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.

  3. Application of an RNA amplification method for reliable single-cell transcriptome analysis

    PubMed Central

    Suslov, Oleg; Silver, Daniel J.; Siebzehnrubl, Florian A.; Orjalo, Arturo; Ptitsyn, Andrey; Steindler, Dennis A.

    2016-01-01

    Diverse cell types have unique transcriptional signatures that are best interrogated at single-cell resolution. Here we describe a novel RNA amplification approach that allows for high fidelity gene profiling of individual cells. This technique significantly diminishes the problem of 3′ bias, enabling detection of all regions of transcripts, including the recognition of mRNA with short or completely absent poly(A) tails, identification of noncoding RNAs, and discovery of the full array of splice isoforms from any given gene product. We assess this technique using statistical and bioinformatics analyses of microarray data to establish the limitations of the method. To demonstrate applicability, we profiled individual cells isolated from the mouse subventricular zone (SVZ)—a well-characterized, discrete yet highly heterogeneous neural structure involved in persistent neurogenesis. Importantly, this method revealed multiple splice variants of key germinal zone gene products within individual cells, as well as an unexpected coexpression of several mRNAs considered markers of distinct and separate SVZ cell types. These findings were independently confirmed using RNA-fluorescence in situ hybridization (RNA-FISH), contributing to the utility of this new technology that offers genomic and transcriptomic analysis of small numbers of dynamic and clinically relevant cells. PMID:26345506

  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. Construction and optimization of an efficient amplification method of a random ssDNA library by asymmetric emulsion PCR.

    PubMed

    Shao, Keke; Shi, Xinhui; Zhu, Xiangjun; Cui, Leilei; Shao, Qixiang; Ma, Da

    2015-12-16

    Construction of a random ssDNA sublibrary is an important step of the aptamer screening process. The available construction methods include asymmetric PCR, biotin-streptavidin separation, and lambda exonuclease digestions, in which PCR amplification is a key step. The main drawback of PCR amplification is overamplification increasing nonspecific hybridization among different products and by-products, which may cause the loss of potential high-quality aptamers, inefficient screening, and even screening failure. Cycle number optimization in PCR amplification is the main way to avoid overamplification but does not fundamentally eliminate the nonspecific hybridization, and the decreased cycle number may lead to insufficient product amounts. Here, we developed a new method, "asymmetric emulsion PCR," which could overcome the shortcomings of conventional PCR. In asymmetric emulsion PCR, different templates were separated by emulsion particles, allowing single-molecule PCR, in which each template was separately amplified, and the nonspecific hybridization was avoided. Overamplification or formation of by-products was not observed. The method is so simple that direct amplification of 40 or more cycles can provide a high-quality ssDNA library. Therefore, the asymmetric emulsion PCR would improve the screening efficiency of systematic evolution of ligands by exponential enrichment.

  6. Novel Degenerate PCR Method for Whole-Genome Amplification Applied to Peru Margin (ODP Leg 201) Subsurface Samples.

    PubMed

    Martino, Amanda J; Rhodes, Matthew E; Biddle, Jennifer F; Brandt, Leah D; Tomsho, Lynn P; House, Christopher H

    2012-01-01

    A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3' end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples.

  7. Loop-mediated isothermal amplification method for differentiation and rapid detection of Taenia species.

    PubMed

    Nkouawa, Agathe; Sako, Yasuhito; Nakao, Minoru; Nakaya, Kazuhiro; Ito, Akira

    2009-01-01

    Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic.

  8. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    PubMed

    Noguchi, Chiemi; Araki, Yoshio; Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  9. The Effect of Storage and Extraction Methods on Amplification of Plasmodium falciparum DNA from Dried Blood Spots.

    PubMed

    Schwartz, Alanna; Baidjoe, Amrish; Rosenthal, Philip J; Dorsey, Grant; Bousema, Teun; Greenhouse, Bryan

    2015-05-01

    Extraction and amplification of DNA from dried blood spots (DBS) collected in field studies is commonly used for detection of Plasmodium falciparum. However, there have been few systematic efforts to determine the effects of storage and extraction methods on the sensitivity of DNA amplification. We investigated the effects of storage conditions, length of storage, and DNA extraction methods on amplification via three PCR-based assays using field samples and laboratory controls. Samples stored as DBS for 2 or more years at ambient temperature showed a significant loss of sensitivity that increased with time; after 10 years only 10% samples with parasite densities > 1,000 parasites/μL were detectable by nested polymerase chain reaction (PCR). Conversely, DBS and extracted DNA stored at -20°C showed no loss of sensitivity with time. Samples with low parasite densities amplified more successfully with saponin/Chelex compared with spin-column-based extraction, though the latter method performed better on samples with higher parasite densities stored for 2 years at ambient temperature. DNA extracted via both methods was stable after 20 freeze-thaw cycles. Our results suggest that DBS should be stored at -20°C or extracted immediately, especially if anticipating 2 or more years of storage.

  10. Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods.

    PubMed

    Lih, Chih-Jian; Si, Han; Das, Biswajit; Harrington, Robin D; Harper, Kneshay N; Sims, David J; McGregor, Paul M; Camalier, Corinne E; Kayserian, Andrew Y; Williams, P Mickey; He, Hua-Jun; Almeida, Jamie L; Lund, Steve P; Choquette, Steve; Cole, Kenneth D

    2016-09-01

    The National Institute of Standards and Technology (NIST) Standard Reference Materials 2373 is a set of genomic DNA samples prepared from five breast cancer cell lines with certified values for the ratio of the HER2 gene copy number to the copy numbers of reference genes determined by real-time quantitative PCR and digital PCR. Targeted-amplicon, whole-exome, and whole-genome sequencing measurements were used with the reference material to compare the performance of both the laboratory steps and the bioinformatic approaches of the different methods using a range of amplification ratios. Although good reproducibility was observed in each next-generation sequencing method, slightly different HER2 copy numbers associated with platform-specific biases were obtained. This study clearly demonstrates the value of Standard Reference Materials 2373 as reference material and as a calibrator for evaluating assay performance as well as for increasing confidence in reporting HER2 amplification for clinical applications.

  11. Analysis of numerical stability and amplification matrices: Fourth-order Runge-Kutta methods

    NASA Technical Reports Server (NTRS)

    Kennedy, E. W.

    1979-01-01

    Amplification matrices, numerical kernels, stable, and exponentially stable numerical solutions are examined. The various techniques involved in these concepts are applied to certain systems that have Jordan forms, which are nondiagonal, with particular interest in the case of imaginary or zero eigenvalues.

  12. 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

  13. Comparison of three DNA extraction methods for feed products and four amplification methods for the 5'-junction fragment of Roundup Ready soybean.

    PubMed

    Wang, Xiumin; Teng, Da; Tian, Fang; Guan, Qingfeng; Wang, Jianhua

    2012-05-09

    Three methods of DNA extraction from feed products and four detection methods for the 5'-junction fragment of genetically modified (GM) Roundup Ready soybean (RRS) were compared and evaluated. The DNA extraction methods, including cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and guanidine hydrochloride (Kit), were assessed for their yields and purity of DNA, extraction time, and reagent cost. The DNA yields of CTAB, SDS, and Kit were 52-694, 164-1750 and 23-105 ng/mg sample, and their extraction time was 2.5-3, 2-2.5, and 1.5-2 h with reagent cost about US dollar 0.24, 0.13, and 1.9 per extraction, respectively. The SDS method was generally well suited to all kinds of feed matrices tested. The limits of detection for the four amplification protocols, including loop-mediated isothermal amplification (LAMP), hyperbranched rolling circle amplification (HRCA), conventional polymerase chain reaction (PCR), and real-time PCR, were 48.5, 4.85, 485, and 9 copies of the pTLH10 plasmid, respectively. The ranked results of the four detection methods were based on multiattribute utility theory as follows (from best to worse): HRCA, LAMP, PCR, and real-time PCR. This comparative evaluation was specifically useful for selection of a highly efficient DNA extraction or amplification method for detecting different GM ingredients.

  14. Microfluidic gradient PCR (MG-PCR): a new method for microfluidic DNA amplification.

    PubMed

    Zhang, Chunsun; Xing, Da

    2010-02-01

    This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the temperature gradient easily spans the range from 97 to 52 degrees Celsius. By making full use of the hot (90-97 degrees Celsius) and cold (60-70 degrees Celsius) regions on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination measurements, and fluorescent melting curve analysis.

  15. 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.

  16. 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.

  17. An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods.

    PubMed

    Chen, Nan; Wang, Wei-Min; Wang, Huan-Ling

    2016-01-13

    A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3' ends of cDNA is performed according to the modified classic 3' RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5' ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5' sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5' and promoter sequences. The 5' end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5' ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness.

  18. An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods

    PubMed Central

    Chen, Nan; Wang, Wei-Min; Wang, Huan-Ling

    2016-01-01

    A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3′ ends of cDNA is performed according to the modified classic 3′ RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5′ ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5′ sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5′ and promoter sequences. The 5′ end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5′ ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness. PMID:26758040

  19. A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

    PubMed Central

    Bigdeli, Saharnaz; Dettloff, Roger O.; Frank, Curtis W.; Davis, Ronald W.; Crosby, Laurel D.

    2015-01-01

    Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk

  20. A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification.

    PubMed

    Bigdeli, Saharnaz; Dettloff, Roger O; Frank, Curtis W; Davis, Ronald W; Crosby, Laurel D

    2015-01-01

    Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk

  1. A novel multiplex isothermal amplification method for rapid detection and identification of viruses

    PubMed Central

    Nyan, Dougbeh-Chris; Swinson, Kevin L.

    2015-01-01

    A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30–60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission. PMID:26643761

  2. Nucleic acid detection system and method for detecting influenza

    DOEpatents

    Cai, Hong; Song, Jian

    2015-03-17

    The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. [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.

  9. 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.

  10. 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.

  11. 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).

  12. 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.

  13. Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification.

    PubMed

    Lenarcic, Erik M; Landry, Dori M; Greco, Todd M; Cristea, Ileana M; Thompson, Sunnie R

    2013-08-01

    Eukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection. Proteins bound to viral RNA are cross-linked prior to cell lysis, purified, and identified using mass spectrometry. Using the TUX-MS method, an unbiased screen for poliovirus (PV) host factors was conducted. All host and viral proteins that are known to interact with the poliovirus RNA were identified. In addition, TUX-MS identified an additional 66 host proteins that have not been previously described in poliovirus amplification. From these candidates, eight were selected and validated. Furthermore, we demonstrate that small interfering RNA (siRNA)-mediated knockdown of two of these uncharacterized host factors results in either a decrease in copy number of positive-stranded RNA or a decrease in PV translation. These data demonstrate that TUX-MS is a robust, unbiased method to identify previously unknown host cell factors that influence virus growth. This method is broadly applicable to a range of RNA viruses, such as flaviviruses, alphaviruses, picornaviruses, bunyaviruses, and coronaviruses.

  14. A novel method of real-time reverse-transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of human astrovirus.

    PubMed

    Wei, Haiyan; Zeng, Jing; Deng, Congliang; Zheng, Chengzhong; Zhang, Ximeng; Ma, Dan; Yi, Yong

    2013-03-01

    A one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) method targeting the 5' end of the capsid gene for rapid and quantitative detection of human astrovirus serotype 1 (HAstV 1) was developed. The assay is highly sensitive and comparable to real-time RT-PCR (rRT-PCR), with a detection limit of ∼100 RNA copies per assay. The specificity of the method was validated by the absence of any cross-reaction with RNA samples of HAstV 2-8 and other gastroenteritis viruses, followed by nucleotide sequencing of the amplified product. Fecal specimens (n=120) obtained from children under five years of age with gastroenteritis were tested by rRT-LAMP, rRT-PCR and transmission electron microscopy (TEM). Six (5%) of these samples were determined to be positive by both rRT-LAMP and rRT-PCR assay, and these two nucleic acid amplification methods resulted in a 200% increase in detection rates for HAstV infection compared with TEM alone. Furthermore, the rRT-LAMP assay is much more rapid than rRT-PCR and generates results in less than 20min for positive samples. The quantitation of viral load in stool specimens was determined from the standard curve plot of time-of-positivity versus initial RNA concentration. Most viral loads were determined to be within the range of 10(5)-10(8) copies. The results highlight the significance of the rapid rRT-LAMP method as a diagnostic and routine screening tool for the analysis of stool samples in hospital laboratories.

  15. Mycoplasma pneumoniae: Current Knowledge on Nucleic Acid Amplification Techniques and Serological Diagnostics

    PubMed Central

    Loens, Katherine; Ieven, Margareta

    2016-01-01

    Mycoplasma pneumoniae (M. pneumoniae) belongs to the class Mollicutes and has been recognized as a common cause of respiratory tract infections (RTIs), including community-acquired pneumonia (CAP), that occur worldwide and in all age groups. In addition, M. pneumoniae can simultaneously or sequentially lead to damage in the nervous system and has been associated with a wide variety of other acute and chronic diseases. During the past 10 years, the proportion of LRTI in children and adults, associated with M. pneumoniae infection has ranged from 0 to more than 50%. This variation is due to the age and the geographic location of the population examined but also due to the diagnostic methods used. The true role of M. pneumoniae in RTIs remains a challenge given the many limitations and lack of standardization of the applied diagnostic tool in most cases, with resultant wide variations in data from different studies. Correct and rapid diagnosis and/or management of M. pneumoniae infections is, however, critical to initiate appropriate antibiotic treatment and is nowadays usually done by PCR and/or serology. Several recent reviews, have summarized current methods for the detection and identification of M. pneumoniae. This review will therefore provide a look at the general principles, advantages, diagnostic value, and limitations of the most currently used detection techniques for the etiological diagnosis of a M. pneumoniae infection as they evolve from research to daily practice. PMID:27064893

  16. Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

    PubMed Central

    2010-01-01

    Background Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic

  17. 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.

  18. Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method.

    PubMed

    Suebsing, Rungkarn; Kampeera, Jantana; Sirithammajak, Sarawut; Pradeep, Padmaja Jayaprasad; Jitrakorn, Sarocha; Arunrut, Narong; Sangsuriya, Pakkakul; Saksmerprome, Vanvimon; Senapin, Saengchan; Withyachumnarnkul, Boonsirm; Kiatpathomchai, Wansika

    2015-12-09

    Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.

  19. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection.

    PubMed

    Nzelu, Chukwunonso O; Gomez, Eduardo A; Cáceres, Abraham G; Sakurai, Tatsuya; Martini-Robles, Luiggi; Uezato, Hiroshi; Mimori, Tatsuyuki; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2014-04-01

    Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)--mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries.

  20. Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk.

    PubMed

    Liu, Y F; Gao, J L; Yang, Y F; Ku, T; Zan, L S

    2014-11-01

    Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 ± 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 μg/μL and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk.

  1. Method for surface plasmon amplification by stimulated emission of radiation (SPASER)

    DOEpatents

    Stockman, Mark I.; Bergman, David J.

    2011-09-13

    A nanostructure is used to generate a highly localized nanoscale optical field. The field is excited using surface plasmon amplification by stimulated emission of radiation (SPASER). The SPASER radiation consists of surface plasmons that undergo stimulated emission, but in contrast to photons can be localized within a nanoscale region. A SPASER can incorporate an active medium formed by two-level emitters, excited by an energy source, such as an optical, electrical, or chemical energy source. The active medium may be quantum dots, which transfer excitation energy by radiationless transitions to a resonant nanosystem that can play the same role as a laser cavity in a conventional laser. The transitions are stimulated by the surface plasmons in the nanostructure, causing the buildup of a macroscopic number of surface plasmons in a single mode.

  2. 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.

  3. 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

  4. Well acidizing compositions and method

    SciTech Connect

    Gardener, T.R.; Dill, W.R.; Ford, W.G.F.; King, K.L.

    1991-07-23

    This patent describes a concentrate which forms an acid internal microemulsion well treatment composition when added to an acid treatment fluid. It comprises in the range of from about 20% to about 98% by weight of a hydrocarbon carrier fluid; in the range of from about 1% to about 50% by weight of an alkyl alcohol having in the range of from about 4 to 18 carbon atoms; and in the range of from about 1% to about 50% by weight of an emulsifying agent comprising at least one compound selected from the group consisting of amine salts having ester or amide linkages and propoxylated alcohols, each of the components being different compounds or different mixtures of compounds.

  5. New method for the visual detection of human respiratory syncytial virus using reverse transcription loop-mediated amplification.

    PubMed

    Mu, Yonglin; Zeng, Jiawei; Chen, Qianqian; Liu, Jia; Wang, Lili; Yao, Fujia; Cui, Meng; He, Zhixiang; Zhang, Chiyu; Xiao, Ming; Lan, Ke

    2014-09-01

    Human respiratory syncytial virus (HRSV) is a seasonal respiratory pathogen that causes respiratory infection in children and the elderly. A new, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid (within 1h), simultaneous detection of A and B group HRSV. Primers specific for groups A and B were designed to amplify the N and L genes of HRSV, respectively. A fluorescent dye, calcein, was used as an indicator for the endpoint visual detection and/or real-time amplification of HRSV RNA. The detection limit of the new method was 281.17 50% tissue culture infective doses (TCID50)/ml for HRSV A and 1.58 TCID50/ml for HRSV B. To evaluate the validity of this method, a comparison with RT-PCR was performed using 77 nasopharyngeal swabs as samples. Both RT-LAMP and RT-PCR detected HRSV in 38 HRSV samples, yielding a positive rate of 49%. Of the RT-LAMP positive samples, 36 (95%) were also positive by RT-PCR, while two were negative by RT-PCR. Among the 36 RT-LAMP and RT-PCR positive samples, 11 belonged to HRSV group A, while 25 belonged to group B. The results show that the new RT-LAMP is simple, rapid and well suited for HRSV diagnosis, especially in a limited-resource setting.

  6. [Evaluation of 3 methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA using PCR].

    PubMed

    Mesquita, R A; Anzai, E K; Oliveira, R N; Nunes, F D

    2001-01-01

    There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

  7. Detection methods for rice viruses by a reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Sasaya, Takahide

    2015-01-01

    Developing a quick and accurate method to diagnose rice viruses in host plants and in vector insects is very important to control virus diseases of rice. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay, one of the most promising molecular diagnostic methods, was established to detect nine viruses, including eight RNA viruses and one DNA virus, in infected rice plants and the viruliferous vector insects. The sensitivities of the assays were either higher than or similar to those of one-step RT-PCR. With a combination of rapid RNA extraction and a RT-LAMP assay, these nine viruses were detected within 2 h from infected rice plants and the viruliferous insects without expensive or unusual equipment. This RT-LAMP method for rice viruses can therefore be adopted not only for diagnosis but also to study the epidemiology and molecular pathology of rice viruses.

  8. Determination of ABO blood group genotypes using the real-time loop-mediated isothermal amplification method

    PubMed Central

    ZHANG, CHAO; ZHU, JUANLI; YANG, JIANGCUN; WAN, YINSHENG; MA, TING; CUI, YALI

    2015-01-01

    ABO genotyping is commonly used in several situations, including blood transfusion, personal identification and disease detection. The present study developed a novel method for ABO genotyping, using loop-mediated isothermal amplification (LAMP). This method allows the simultaneous determination of six ABO genotypes under 40 min at a constant temperature of 62°C. The genotypes of 101 blood samples were determined to be AA (n=6), AO (n=38), BB (n=12), BO (n =29), AB (n=8) and OO (n=8) by the LAMP assay. The results were compared with the phenotypes determined by serological assay and the genotypes determined by direct sequencing, and no discrepancies were observed. This novel and rapid method, with good accuracy and reasonably cost effective, provides a supplement to routine serological ABO typing and may also be useful in other point-of-care testing. PMID:26238310

  9. 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.

  10. An efficient and high fidelity method for amplification, cloning and sequencing of complete tospovirus genomic RNA segments.

    PubMed

    Marshall, Spencer H; Adegbola, Raphael O; Adkins, Scott; Naidu, Rayapati A

    2017-04-01

    Tospoviruses (genus Tospovirus, family Bunyaviridae) are responsible for major losses in an extensive range of crops worldwide. New species of these single-stranded, ambisense RNA viruses regularly emerge and have been shown to maintain heterogeneous populations with individual isolates having quite variable biological and virulence characteristics. Most tospovirus phylogenetic studies have focused on analysis of a single gene, most often the nucleocapsid protein gene. Complete genomic RNA segment amplification as a single fragment would facilitate more detailed analyses of genome-wide sequence variability, but obtaining such sequences for a large number of tospovirus isolates using traditional methods of amplification and cloning of small overlapping fragments is tedious, time consuming and expensive. In this study, protocols were optimized to amplify, clone and sequence full-length M- and S-RNA genome segments of Tomato spotted wilt virus and Impatiens necrotic spot virus. The strategy presented here is straightforward, scalable and offers several advantages over the previously commonplace and overlapping amplicon-based approach. Use of whole genome segments, instead of individual gene sequences or defined portions of genome segments, will facilitate a better understanding of the underlying molecular diversity of tospoviruses in mixed infections.

  11. Effect of cohesion and fill amplification on seismic stability of municipal solid waste landfills using limit equilibrium method.

    PubMed

    Savoikar, Purnanand; Choudhury, Deepankar

    2010-12-01

    Municipal solid waste (MSW) landfills in seismic zones are subjected to the seismic forces both in the horizontal and vertical directions. The stability of landfills against these seismic forces was evaluated by computing the factor of safety of landfills with different modes of failure among which failures of landfills due to translation are very common. Conventionally, the seismic stability of landfill is evaluated by using pseudo-static limit equilibrium method. In the present study, seismic stability of landfills is evaluated by both the conventional pseudo-static and modern pseudo-dynamic method. The pseudo-dynamic method is superior as it takes into account the effect of duration and frequency of earthquake motion and corresponding body waves in addition to the variation of earthquake accelerations along depth and time. In the present study, the effects of cohesion and fill amplification on seismic stability of landfill are also taken into account. It was noticed that, neglecting cohesion of fill material as well as liner material, results in a lower factor of safety and, hence, a very conservative/uneconomic design. Also, fill amplification is found to reduce the factor of safety values computed only by using the pseudo-dynamic method, showing its advantage. Generalized expressions are developed for factor of safety and yield acceleration against translational failure, which can be used for evaluating the seismic stability of MSW landfills. Comparisons of results under static condition with existing, similar methodology show a very good agreement. However, the present study seems to provide unique results for the seismic case.

  12. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-05-30

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  13. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-06-06

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  14. Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

    PubMed

    Rohrman, Brittany; Richards-Kortum, Rebecca

    2015-02-03

    Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads.

  15. Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas

    PubMed Central

    Chen, Xiaolu; Ma, Lijuan; Qiang, Song; Ma, Deying

    2016-01-01

    Ascochyta blight (AB) is a devastating fungal disease of chickpeas that has spread to nearly all of the chickpea cultivating regions of the world. The rapid diagnosis of Ascochyta rabiei L. (A. rabiei), the pathogen that causes AB, plays an important role in A. rabiei epidemic tracking and AB management. In this study, a group of loop-mediated isothermal amplification (LAMP) primers was designed to detect A. rabiei in chickpea plants and seeds via a LAMP method and a conventional PCR method based on an internal transcribed spacer (ITS) sequence analysis of A. rabiei. Compared with the conventional PCR method, the LAMP method not only exhibited greater sensitivity and specificity in the detection of A. rabiei but also used simpler equipment and required less operational time. The minimum detectable concentration of the A. rabiei genomic DNA solution with the LAMP method was 6.01 × 10−6 ng/μl, which was 100 times lower than that of the conventional PCR method with the same outer primers. The greatest advantage of the LAMP method is that results can be observed via the visualization of color changes in SYBR Green I dye with the naked eye, and it does not require expensive instruments, also with less time consumption. PMID:27161564

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B.

    PubMed

    Cao, Cuiming; Zhang, Fang; Ji, Mingyu; Pei, Fengyan; Fan, Xiujie; Shen, Hong; Wang, Qingxi; Yang, Weihua; Wang, Yunshan

    2016-07-01

    We developed a visual loop-mediated isothermal amplification (LAMP) technique to detect the streptococcal pyrogenic exotoxin B (speB) gene. Fifteen strains (from American Type Culture Collection or clinical isolates) were used to determine the specificity and sensitivity of the LAMP assay. Clinical samples were collected from 132 patients with suspected Streptococcus pyogenes (S. pyogenes) infection to verify the feasibility of the LAMP assay for detection of the speB gene. By using a set of five primers (a pair of outer primers, a pair of inner primers and one loop primer) targeting the speB gene, the amplification reaction was rapidly performed in a regular water bath under isothermal conditions at 63 °C for approximately 60 min. Only the two S. pyogenes strains showed positive results which were easily observed with the naked eye, and the other strains showed negative results. The detection limit of the LAMP assay was 0.01 ng/μl of template, showing higher sensitivity than conventional PCR (with a detection limit of 1.0 ng/μl). The detection rate of the speB gene in clinical samples was 71.21% and was consistent with the PCR results. The rapid detection of the speB gene by the LAMP assay is highly specific and sensitive, is simple to perform and cost-effective, and is expected to be a new reliable method for the rapid diagnosis of S. pyogenes infection, that is particularly suitable for rural or community hospitals in developing countries.

  2. Comparison of in situ hybridization methods for the assessment of HER-2/neu gene amplification status in breast cancer using a tissue microarray

    PubMed Central

    Malicka-Durczak, Anna; Korski, Konstanty; Ibbs, Matthew

    2012-01-01

    Background This project compared HER-2/neu gene status in breast cancers, as demonstrated by FISH (fluorescent in situ hybridization) and CISH (chromogenic in situ hybridization) and using a tissue microarray (TMA). The study also aimed to show whether the TMA technique could be used in clinical diagnostics, rather than remain a scientific tool. Materials and methods A TMA was constructed using 121 breast cancer specimens, 6 cores from each specimen. Demonstration and assessment of HER-2/neu gene status was by FISH (Vysis Path) and CISH (DAKO Duo CISH). Results The 121 breast cancer specimens were divided into 3 groups by HER-2 status, as determined by immunohistochemistry. In the HER-2 negative group no amplification was observed in 36 out of 40 cases. 3 cases showed amplification by both methods and one by CISH alone. The equivocal HER-2 group showed no amplification in 30 out of 41 cases and amplification in 9 cases. One case was FISH negative CISH positive and one was discarded. In the HER-2 positive group, amplification was confirmed in 37 of the 40 cases by both methods. 3 cases were unsuitable for assessment. Conclusions This study indicated that CISH is a sensitive alternative to FISH in detecting HER2 gene amplification and may replace FISH in HER2 testing. Good agreement was observed between methods (98.5% – 119 out of 121 cases). Furthermore, as only 4 out of 121 cases were unsuitable for assessment (no signal or missing TMA cores) – it may be feasible to use TMA in diagnostics. PMID:24377000

  3. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China

    PubMed Central

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 102 CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient. PMID:28197157

  4. A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes.

    PubMed

    Shi, Xianzong; Jarvis, Donald L

    2006-09-15

    Rapid amplification of cDNA ends (RACE) is widely used to determine the 5'- and 3'-terminal nucleotide sequences of genes. Many different RACE methods have been developed to meet various requirements, but none addresses the difficult problems that arise when trying to isolate the ends of extremely guanine plus cytosine (GC)-rich genes. In this study, we found that we were unable to isolate the correct 5' or 3' end of an insect gene, which appeared to include extremely GC-rich sequences, using current RACE methods. Thus, we developed a new RACE method that can be used for this purpose. This new method entails first-strand cDNA synthesis at 70 degrees C with Thermo-X reverse transcriptase in the presence of homoectoine, followed by a polymerase chain reaction with 98 degrees C denaturation steps and Phusion DNA polymerase in the presence of 1M betaine and 5% dimethyl sulfoxide (DMSO). The use of these conditions yielded 5'- and 3'-RACE products that were approximately 80% GC over 213 and 162bp, respectively, and included shorter internal regions of 82 to 89% GC.

  5. Modeling and Calculation of Optical Amplification in One Dimensional Case of Laser Medium Using Finite Difference Time Domain Method

    NASA Astrophysics Data System (ADS)

    Maryana, Okky Fajar Tri; Hidayat, Rahmat

    2016-08-01

    Finite Difference Time Domain (FDTD) method has been much employed for studying light propagation in various structures, from simple one-dimensional structures up to three-dimensional complex structures. One of challenging problems is to implement this method for the case of light propagation in amplifying medium or structures, such as optical amplifier and lasers. The implementation is hindered by the fact that the dielectric constant becomes a complex number when optical gain parameter is involved in the calculation. In general, complex dielectric constant is related to complex susceptibility, in which the imaginary part is related to optical gain. Here, we then modify the formulation for updating electric field in the calculation algorithm. Using this approach, we then finally can calculate light amplification in laser active medium of Nd3+ ion doped glass. The calculation result shows an agreement with the result from the calculation using differential equation for intensity. Although this method is more time consuming, the method seem promising for optical complex micro- and nano-structures, such quantum dot lasers, micro-ring lasers, etc.

  6. Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method.

    PubMed

    Honda, Shozo; Morichika, Keisuke; Kirino, Yohei

    2016-03-01

    RNA digestions catalyzed by many ribonucleases generate RNA fragments that contain a 2',3'-cyclic phosphate (cP) at their 3' termini. However, standard RNA-seq methods are unable to accurately capture cP-containing RNAs because the cP inhibits the adapter ligation reaction. We recently developed a method named cP-RNA-seq that is able to selectively amplify and sequence cP-containing RNAs. Here we describe the cP-RNA-seq protocol in which the 3' termini of all RNAs, except those containing a cP, are cleaved through a periodate treatment after phosphatase treatment; hence, subsequent adapter ligation and cDNA amplification steps are exclusively applied to cP-containing RNAs. cP-RNA-seq takes ∼6 d, excluding the time required for sequencing and bioinformatics analyses, which are not covered in detail in this protocol. Biochemical validation of the existence of cP in the identified RNAs takes ∼3 d. Even though the cP-RNA-seq method was developed to identify angiogenin-generating 5'-tRNA halves as a proof of principle, the method should be applicable to global identification of cP-containing RNA repertoires in various transcriptomes.

  7. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China.

    PubMed

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 10(2) CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient.

  8. Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method

    PubMed Central

    Honda, Shozo; Morichika, Keisuke; Kirino, Yohei

    2016-01-01

    RNA digestions catalyzed by many ribonucleases generate RNA fragments containing a 2′,3′-cyclic phosphate (cP) at their 3′-termini. However, standard RNA-seq methods are unable to accurately capture cP-containing RNAs because the cP inhibits the adapter ligation reaction. We recently developed a method named “cP-RNA-seq” that is able to selectively amplify and sequence cP-containing RNAs. Here we describe the cP-RNA-seq protocol in which the 3′-termini of all RNAs, except those containing a cP, are cleaved through a periodate treatment after phosphatase treatment, hence subsequent adapter ligation and cDNA amplification steps are exclusively applied to cP-containing RNAs. cP-RNA-seq takes ~6 d, excluding the time required for sequencing and bioinformatics analyses, such downstream assays are not covered in detail in this protocol. Biochemical validation of the existence of cP in the identified RNAs takes ~3 d. Even though the cP-RNA-seq method was developed to identify angiogenin-generating 5′-tRNA halves as a proof of principle, the method should be applicable to global identification of cP-containing RNA repertoires in various transcriptomes. PMID:26866791

  9. Methods for analyzing nucleic acid sequences

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2011-05-17

    The present invention is directed to a method of sequencing a target nucleic acid. The method provides a complex comprising a polymerase enzyme, a target nucleic acid molecule, and a primer, wherein the complex is immobilized on a support Fluorescent label is attached to a terminal phosphate group of the nucleotide or nucleotide analog. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The time duration of the signal from labeled nucleotides or nucleotide analogs that become incorporated is distinguished from freely diffusing labels by a longer retention in the observation volume for the nucleotides or nucleotide analogs that become incorporated than for the freely diffusing labels.

  10. Phenolic acid esterases, coding sequences and methods

    DOEpatents

    Blum, David L.; Kataeva, Irina; Li, Xin-Liang; Ljungdahl, Lars G.

    2002-01-01

    Described herein are four phenolic acid esterases, three of which correspond to domains of previously unknown function within bacterial xylanases, from XynY and XynZ of Clostridium thermocellum and from a xylanase of Ruminococcus. The fourth specifically exemplified xylanase is a protein encoded within the genome of Orpinomyces PC-2. The amino acids of these polypeptides and nucleotide sequences encoding them are provided. Recombinant host cells, expression vectors and methods for the recombinant production of phenolic acid esterases are also provided.

  11. Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.

    PubMed

    Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2014-06-01

    Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests.

  12. Diagnostic value of the strand displacement amplification method compared to those of Roche Amplicor PCR and culture for detecting mycobacteria in sputum samples.

    PubMed Central

    Ichiyama, S; Ito, Y; Sugiura, F; Iinuma, Y; Yamori, S; Shimojima, M; Hasegawa, Y; Shimokata, K; Nakashima, N

    1997-01-01

    We compared the ability of the semiautomated BDProbeTec-SDA system, which uses the strand displacement amplification (SDA) method, with that of the Roche Amplicor-PCR system and the Septi-Chek AFB culture system to directly detect Mycobacterium tuberculosis complex (MTB) and other mycobacteria in sputum samples. A total of 530 sputum samples from 299 patients were examined in this study. Of the 530 samples, 129 were culture positive for acid-fast bacilli with the Septi-Chek AFB system; 95 for MTB, 29 for M. avium-M. intracellulare complex (MAC), and 5 for other mycobacteria. The BDProbeTec-SDA system detected 90 of the 95 samples culture positive for MTB (sensitivity, 94.7%), and the Amplicor-PCR system detected 85 of the 95 samples culture positive for MTB (sensitivity, 89.5%). The specificity of each system, based on the clinical diagnosis, was 99.8% for SDA and 100% for PCR, respectively. Among the 29 samples culture positive for MAC, the BDProbeTec-SDA system detected MAC in 24 samples (sensitivity, 82.8%), whereas the Amplicor-PCR system detected MAC in 23 samples (sensitivity, 79.3%). The specificities of the systems were 98.3 and 100%, respectively. The high degrees of sensitivity and specificity of the BDProbeTec-SDA system suggest that it should be very useful in clinical laboratories for the rapid detection of mycobacteria in sputum samples. PMID:9399498

  13. Detection of avian retroviruses in vaccines by amplification on DF-1 cells with immunostaining and fluorescent product-enhanced reverse transcriptase endpoint methods.

    PubMed

    Birmingham, Cheryl L; Dupont, Dominique; Riou, Patrice; Armanet, Corinne; Edamura, Kerrie Nichol; Martinho, Briolange; Serres, Aurelie; Jacouton, Severine; Detrez, Valerie; McNeil, Bryan; Schreiber, Martha; Gaillac, David; Bonnevay, Thierry; Gisonni-Lex, Lucy; Mallet, Laurent

    2013-05-01

    In order to ensure the safety of vaccines produced on avian cells, rigorous testing for the absence of avian retroviruses must be performed. Current methods used to detect avian retroviruses often exhibit a high invalid-test/false-positive rate, rely on hard-to-secure reagents, and/or have readouts that are difficult to standardize. Herein, we describe the development and validation of two consistent and sensitive methods for the detection of avian retroviruses in vaccines: viral amplification on DF-1 cells followed by immunostaining for the detection of avian leukosis virus (ALV) and viral amplification on DF-1 cells followed by fluorescent product-enhanced reverse transcriptase (F-PERT) for the detection of all avian retroviruses. Both assays share an infectivity stage on DF-1 cells followed by a different endpoint readout depending on the retrovirus to be detected. Validation studies demonstrated a limit of detection of one 50% cell culture infectious dose (CCID(50))/ml for retrovirus in a 30-ml test inoculum volume for both methods, which was as sensitive as a classical method used in the vaccine industry, namely, viral amplification on primary chicken embryo fibroblasts followed by the complement fixation test for avian leukosis virus (COFAL). Furthermore, viral amplification on DF-1 cells followed by either immunostaining or F-PERT demonstrated a sensitivity that exceeds the regulatory requirements for detection of ALV strains. A head-to-head comparison of the two endpoint methods showed that viral amplification on DF-1 cells followed by F-PERT is a suitable method to be used as a stand-alone test to ensure that vaccine preparations are free from infectious avian retroviruses.

  14. Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods.

    PubMed

    Jara, C; Mateo, E; Guillamón, J M; Torija, M J; Mas, A

    2008-12-10

    Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery.

  15. Modified PCR methods for 3' end amplification from serial analysis of gene expression (SAGE) tags.

    PubMed

    Xu, Wang-Jie; Wang, Zhao-Xia; Qiao, Zhong-Dong

    2009-05-01

    Serial analysis of gene expression (SAGE) is a powerful technique to study gene expression at the genome level. However, a disadvantage of the shortness of SAGE tags is that it prevents further study of SAGE library data, thus limiting extensive application of the SAGE method in gene expression studies. However, this problem can be solved by extension of the SAGE tags to 3' cDNAs. Therefore, several methods based on PCR have been developed to generate a 3' longer fragment cDNA corresponding to a SAGE tag. The list of modified methods is extensive, and includes rapid RT-PCR analysis of unknown SAGE tags (RAST-PCR), generation of longer cDNA fragments from SAGE tags for gene identification (GLGI), a high-throughput GLGI procedure, reverse SAGE (rSAGE), two-step analysis of unknown SAGE tags (TSAT-PCR), etc. These procedures are constantly being updated because they have characteristics and advantages that can be shared. Development of these methods has promoted the widespread use of the SAGE technique, and has accelerated the speed of studies of large-scale gene expression.

  16. Highly Sensitive Detection of Low-Abundance White Spot Syndrome Virus by a Pre-Amplification PCR Method.

    PubMed

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

    2017-03-28

    White spot syndrome virus (WSSV) is a major threat to the shrimp farming industry and so far there is no effective therapy for it, and thus early diagnostic of WSSV is of great importance. However, at the early stage of infection, the extremely low-abundance of WSSV DNA challenges the detection sensitivity and accuracy of PCR. To effectively detect low-abundance WSSV, here we developed a pre-amplification PCR (pre-amp PCR) method to amplify trace amounts of WSSV DNA from massive background genomic DNA. Combining with normal specific PCR, 10 copies of target WSSV genes were detected from ~10(10) magnitude of backgrounds. In particular, multiple target genes were able to be balanced amplified with similar efficiency due to the usage of the universal primer. The efficiency of the pre-amp PCR was validated by nested-PCR and quantitative PCR, and pre-amp PCR showed higher efficiency than nested-PCR when multiple targets were detected. The developed method is particularly suitable for the super early diagnosis of WSSV, and has potential to be applied in other low-abundance sample detection cases.

  17. The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA.

    PubMed

    Billard, A; Laval, V; Fillinger, S; Leroux, P; Lachaise, H; Beffa, R; Debieu, D

    2012-02-01

    The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

  18. PCR-sequencing is a complementary method to amplification refractory mutation system for EGFR gene mutation analysis in FFPE samples.

    PubMed

    Jiang, Junchang; Wang, Chunhua; Yu, Xiaoli; Sheng, Danli; Zuo, Chen; Ren, Minpu; Wu, Yaqin; Shen, Jie; Jin, Mei; Xu, Songxiao

    2015-12-01

    Amplification Refractory Mutation System (ARMS) is the most popular technology for EGFR gene mutation analysis in China. Cutoff Ct or ΔCt values were used to differentiate low mutation abundance cases from no mutation cases. In this study, all of 359 NSCLC samples were tested by ARMS. Seventeen samples with larger Ct or ΔCt than cutoff values were retested by PCR-sequencing. TKI treatment responses were monitored on the cases with ARMS negative and PCR-sequencing positive results. One exon 18 G719X case, 67 exon 19 deletion cases, 2 exon 20 insertion cases, 1 exon 20 T790M case, 60 exon 21 L858R cases, 5 exon 21 L861Q cases and 201 wild type cases were identified by ARMS. Another 22 cases were evaluated as wild type but had later amplification fluorescent curves. Seventeen out of these 22 cases were retested by PCR-sequencing. It turns out that 3 out of 3 cases with exon 19 deletion later amplifications, 2 out of 2 cases with L858R later amplifications and 4 out of 12 cases with T790M later amplifications were identified as mutation positive. Two cases with exon 19 deletion and L858R respectively were treated by TKI and got responses. Our study indicated that PCR-sequencing might be a complementary way to confirm ARMS results with later amplifications.

  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. Loop-mediated isothermal amplification: rapid visual and real-time methods for detection of genetically modified crops.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika; Morisset, Dany; Sood, Payal; Zel, Jana

    2013-11-27

    A rapid, reliable, and sensitive loop-mediated isothermal amplification (LAMP) system was developed for screening of genetically modified organisms (GMOs). The optimized LAMP assays using designed primers target commonly employed promoters, i.e., Cauliflower Mosaic Virus 35S (P-35S) and Figwort Mosaic Virus promoter (P-FMV), and marker genes, i.e., aminoglycoside 3'-adenyltransferase (aadA), neomycin phosphotransferase II (nptII), and β-glucuronidase (uidA). The specificity and performance of the end-point and real-time LAMP assays were confirmed using eight genetically modified (GM) cotton events on four detection systems, employing two chemistries. LAMP assays on the isothermal real-time system were found to be most sensitive, detecting up to four target copies, within 35 min. The LAMP assays herein presented using alternate detection systems can be effectively utilized for rapid and cost-effective screening of the GM status of a sample, irrespective of the crop species or GM trait. These assays coupled with a fast and simple DNA extraction method may further facilitate on-site GMO screening.

  4. Development of a reverse-transcription loop-mediated isothermal amplification method for detection of rabbit hemorrhagic disease virus.

    PubMed

    Yuan, Dongwei; Guo, Dongchun; Liu, Jiasen; Si, Changde; Jiang, Qian; Lin, Huan; Yang, Tiankuo; Qu, Liandong

    2013-02-01

    Rabbit hemorrhagic disease virus (RHDV) causes haemagglutination and severe liver damage, with a high mortality rate. To develop a rapid and sensitive method for the surveillance of RHDV, a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was established using a set of four primers specific for the VP60 gene segment of RHDV. The established assay was performed at 64°C for 40 min under isothermal conditions, and the results were visualized directly by electrophoresis or as fluorescent signals under ultraviolet light. The detection limit of the RT-LAMP assay was 10 copies of viral RNA per reaction, which was comparable to quantitative real-time RT-PCR, and 100-fold more sensitive than standard RT-PCR. Furthermore, seven viral RNAs of field isolates in China could be detected successfully using this assay. Overall, the newly established RT-LAMP assay indicates the potential usefulness of the technique as a simple, rapid and sensitive procedure, and can visually detect RHDV infection without the need for any specialized equipment.

  5. Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing.

    PubMed

    Imai, Kazuo; Tarumoto, Norihito; Amo, Kiyoko; Takahashi, Makoto; Sakamoto, Naoya; Kosaka, Atsushi; Kato, Yasuyuki; Mikita, Kei; Sakai, Jun; Murakami, Takashi; Suzuki, Yutaka; Maesaki, Shigefumi; Maeda, Takuya

    2017-03-11

    Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION™ sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.

  6. Cavitation during the protein misfolding cyclic amplification (PMCA) method – The trigger for de novo prion generation?

    SciTech Connect

    Haigh, Cathryn L.; Drew, Simon C.

    2015-06-05

    The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo. - Highlights: • Sonication during PMCA generates free radicals at the surface of cavitation bubbles. • PrP-centered and RNA-centered radicals are formed in addition to PrP-RNA adducts. • De novo prions may result from ROS and structural constraints during cavitation.

  7. 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.

  8. 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.

  9. A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation

    PubMed Central

    Frithiof, Henrik; Aaltonen, Kristina; Rydén, Lisa

    2016-01-01

    Introduction Amplification of the HER-2/neu (HER-2) proto-oncogene occurs in 10%–15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples. Methods Using healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4′,6-diamidino-2′-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of HER-2 amplification status in breast cancer CTCs. Results A method for defining the presence of HER-2 amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTC HER-2 amplification during treatment against metastatic disease. Conclusion HER-2 amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to

  10. Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA.

    PubMed

    Gupta, Vikas; Chakravarti, Soumendu; Chander, Vishal; Majumder, Saurabh; Bhat, Shabir Ahmad; Gupta, Vivek Kumar; Nandi, Sukdeb

    2017-03-27

    Canine parvovirus-2 (CPV-2), which is ubiquitously distributed worldwide, causes severe and often fatal gastroenteritis in dogs. Accurate, differential and rapid diagnosis of canine parvoviral enteritis remains a challenge for clinicians. A recently developed isothermal amplification technique, polymerase spiral reaction (PSR), was optimized for the first time for a viral pathogen with reference recombinant plasmid standards from different CPV-2 antigenic variants (CPV-2, CPV-2a, CPV-2b and CPV-2c) and subsequently validated using clinical samples. Addition of chromogenic substrate SYBR Green I after the completion of the reaction resulted in bright green fluorescence in positive samples, while negative samples and a no-template control remained orange. These results were further substantiated through visualization of a laddering pattern of the PSR-amplified product in an agarose gel in positive cases and the absence of this pattern in no-template control and negative samples. The PSR assay was found to be highly specific, as it did not react with other putative canine pathogens (canine adenovirus 1 and canine distemper virus). The sensitivity of the newly developed PSR technique was compared with that of conventional PCR, real-time PCR and LAMP, using a serial tenfold dilution of canine parvovirus DNA. The detection limit of PSR was found to be at the femtogram level, which is comparable with that of real-time PCR and LAMP, which are ten times more sensitive than conventional PCR. The assay was validated using 90 clinical samples, of which 54 were found positive, while only 45 samples were positive in conventional PCR. This novel assay, which is fully compliant with the 'ASSURED' concept for disease diagnosis, provides a simple, rapid, specific, sensitive and cost-effective method for diagnosis of canine parvoviral enteritis in veterinary clinics.

  11. Method for the production of dicarboxylic acids

    DOEpatents

    Nghiem, Nhuan Phu; Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1999-01-01

    The present invention is an economical fermentation method for the production of carboxylic acids comprising the steps of a) inoculating a medium having a carbon source with a carboxylic acid-producing organism; b) incubating the carboxylic acid-producing organism in an aerobic atmosphere to promote rapid growth of the organism thereby increasing the biomass of the organism; c) controllably releasing oxygen to maintain the aerobic atmosphere; d) controllably feeding the organism having increased biomass with a solution containing the carbon source to maintain the concentration of the carbon source within the medium of about 0.5 g/L up to about 1 g/L; e) depriving the aerobic atmosphere of oxygen to produce an anaerobic atmosphere to cause the organism to undergo anaerobic metabolism; f) controllably feeding the organism having increased biomass a solution containing the carbon source to maintain the concentration of the carbon source within the medium of .gtoreq.1 g/L; and g) converting the carbon source to carboxylic acids using the anaerobic metabolism of the organism.

  12. Method for the production of dicarboxylic acids

    DOEpatents

    Nghiem, N.P.; Donnelly, M.; Millard, C.S.; Stols, L.

    1999-02-09

    The present invention is an economical fermentation method for the production of carboxylic acids comprising the steps of (a) inoculating a medium having a carbon source with a carboxylic acid-producing organism; (b) incubating the carboxylic acid-producing organism in an aerobic atmosphere to promote rapid growth of the organism thereby increasing the biomass of the organism; (c) controllably releasing oxygen to maintain the aerobic atmosphere; (d) controllably feeding the organism having increased biomass with a solution containing the carbon source to maintain the concentration of the carbon source within the medium of about 0.5 g/l up to about 1 g/l; (e) depriving the aerobic atmosphere of oxygen to produce an anaerobic atmosphere to cause the organism to undergo anaerobic metabolism; (f) controllably feeding the organism having increased biomass a solution containing the carbon source to maintain the concentration of the carbon source within the medium of {>=}1 g/l; and (g) converting the carbon source to carboxylic acids using the anaerobic metabolism of the organism. 7 figs.

  13. 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

  14. TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol

    PubMed Central

    Picher, Ángel J.; Budeus, Bettina; Wafzig, Oliver; Krüger, Carola; García-Gómez, Sara; Martínez-Jiménez, María I.; Díaz-Talavera, Alberto; Weber, Daniela; Blanco, Luis; Schneider, Armin

    2016-01-01

    Sequencing of a single-cell genome requires DNA amplification, a process prone to introducing bias and errors into the amplified genome. Here we introduce a novel multiple displacement amplification (MDA) method based on the unique DNA primase features of Thermus thermophilus (Tth) PrimPol. TthPrimPol displays a potent primase activity preferring dNTPs as substrates unlike conventional primases. A combination of TthPrimPol's unique ability to synthesize DNA primers with the highly processive Phi29 DNA polymerase (Φ29DNApol) enables near-complete whole genome amplification from single cells. This novel method demonstrates superior breadth and evenness of genome coverage, high reproducibility, excellent single-nucleotide variant (SNV) detection rates with low allelic dropout (ADO) and low chimera formation as exemplified by sequencing HEK293 cells. Moreover, copy number variant (CNV) calling yields superior results compared with random primer-based MDA methods. The advantages of this method, which we named TruePrime, promise to facilitate and improve single-cell genomic analysis. PMID:27897270

  15. 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

  16. Development of reverse transcription loop-mediated isothermal amplification assay as a simple detection method of Chrysanthemum stem necrosis virus in chrysanthemum and tomato.

    PubMed

    Suzuki, Ryoji; Fukuta, Shiro; Matsumoto, Yuho; Hasegawa, Toru; Kojima, Hiroko; Hotta, Makiko; Miyake, Noriyuki

    2016-10-01

    For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection.

  17. A Simple Method for the Extraction, PCR-amplification, Cloning, and Sequencing of Pasteuria 16S rDNA from Small Numbers of Endospores

    PubMed Central

    Atibalentja, N.; Noel, G. R.; Ciancio, A.

    2004-01-01

    For many years the taxonomy of the genus Pasteuria has been marred with confusion because the bacterium could not be cultured in vitro and, therefore, descriptions were based solely on morphological, developmental, and pathological characteristics. The current study sought to devise a simple method for PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA from small numbers of endospores, with no need for prior DNA purification. Results show that DNA extracts from plain glass bead-beating of crude suspensions containing 10,000 endospores at 0.2 × 10⁶ endospores ml-1 were sufficient for PCR-amplification of Pasteuria 16S rDNA, when used in conjunction with specific primers. These results imply that for P. penetrans and P. nishizawae only one parasitized female of Meloidogyne spp. and Heterodera glycines, respectively, should be sufficient, and as few as eight cadavers of Belonolaimus longicaudatus with an average number of 1,250 endospores of "Candidatus Pasteuria usgae" are needed for PCR-amplification of Pasteuria 16S rDNA. The method described in this paper should facilitate the sequencing of the 16S rDNA of the many Pasteuria isolates that have been reported on nematodes and, consequently, expedite the classification of those isolates through comparative sequence analysis. PMID:19262793

  18. T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids

    PubMed Central

    Nai, Yu-Shin; Chen, Tzu-Han; Huang, Yu-Feng; Midha, Mohit K.; Shiau, Hsin-Chieh; Shen, Chen-Yang; Chen, Chien-Jen; Yu, Alice L.; Chiu, Kuo Ping

    2017-01-01

    Body fluid DNA sequencing is a powerful noninvasive approach for the diagnosis of genetic defects, infectious agents and diseases. The success relies on the quantity and quality of the DNA samples. However, numerous clinical samples are either at low quantity or of poor quality due to various reasons. To overcome these problems, we have developed T oligo-primed polymerase chain reaction (TOP-PCR) for full-length nonselective amplification of minute quantity of DNA fragments. TOP-PCR adopts homogeneous “half adaptor” (HA), generated by annealing P oligo (carrying a phosphate group at the 5′ end) and T oligo (carrying a T-tail at the 3′ end), for efficient ligation to target DNA and subsequent PCR amplification primed by the T oligo alone. Using DNA samples from body fluids, we demonstrate that TOP-PCR recovers minute DNA fragments and maintains the DNA size profile, while enhancing the major molecular populations. Our results also showed that TOP-PCR is a superior method for detecting apoptosis and outperforms the method adopted by Illumina for DNA amplification. PMID:28094343

  19. T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids.

    PubMed

    Nai, Yu-Shin; Chen, Tzu-Han; Huang, Yu-Feng; Midha, Mohit K; Shiau, Hsin-Chieh; Shen, Chen-Yang; Chen, Chien-Jen; Yu, Alice L; Chiu, Kuo Ping

    2017-01-17

    Body fluid DNA sequencing is a powerful noninvasive approach for the diagnosis of genetic defects, infectious agents and diseases. The success relies on the quantity and quality of the DNA samples. However, numerous clinical samples are either at low quantity or of poor quality due to various reasons. To overcome these problems, we have developed T oligo-primed polymerase chain reaction (TOP-PCR) for full-length nonselective amplification of minute quantity of DNA fragments. TOP-PCR adopts homogeneous "half adaptor" (HA), generated by annealing P oligo (carrying a phosphate group at the 5' end) and T oligo (carrying a T-tail at the 3' end), for efficient ligation to target DNA and subsequent PCR amplification primed by the T oligo alone. Using DNA samples from body fluids, we demonstrate that TOP-PCR recovers minute DNA fragments and maintains the DNA size profile, while enhancing the major molecular populations. Our results also showed that TOP-PCR is a superior method for detecting apoptosis and outperforms the method adopted by Illumina for DNA amplification.

  20. 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.

  1. A loop-mediated isothermal amplification method for a differential identification of Taenia tapeworms from human: application to a field survey.

    PubMed

    Nkouawa, Agathe; Sako, Yasuhito; Li, Tiaoying; Chen, Xingwang; Nakao, Minoru; Yanagida, Tetsuya; Okamoto, Munehiro; Giraudoux, Patrick; Raoul, Francis; Nakaya, Kazuhiro; Xiao, Ning; Qiu, Jiamin; Qiu, Dongchuan; Craig, Philip S; Ito, Akira

    2012-12-01

    In this study, we applied a loop-mediated isothermal amplification method for identification of human Taenia tapeworms in Tibetan communities in Sichuan, China. Out of 51 proglottids recovered from 35 carriers, 9, 1, and 41 samples were identified as Taenia solium, Taenia asiatica and Taenia saginata, respectively. Same results were obtained afterwards in the laboratory, except one sample. These results demonstrated that the LAMP method enabled rapid identification of parasites in the field surveys, which suggested that this method would contribute to the control of Taenia infections in endemic areas.

  2. Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus

    PubMed Central

    Liu, Ningwei; Zou, Dayang; Dong, Derong; Yang, Zhan; Ao, Da; Liu, Wei; Huang, Liuyu

    2017-01-01

    Rapid detection of food-borne pathogens is important in the food industry, to monitor and prevent the spread of these pathogens through contaminated food products. We therefore established a multiplex real-time loop-mediated isothermal amplification (LAMP) assay to simultaneously detect and distinguish Salmonella spp. and Vibrio parahaemolyticus DNA in a single reaction. Two target sequences, one specific for Salmonella and the other specific for Vibrio parahaemolyticus, were amplified by specific LAMP primers in the same reaction tube. After amplification at 65 °C for 60 min, the amplified products were subjected to melting curve analysis and thus could be distinguished based on the different melting temperatures (Tm values) of the two specifically amplified products. The specificity of the multiplex LAMP assay was evaluated using 19 known bacterial strains, including one V. parahaemolyticus and seven Salmonella spp. strains. The multiplex LAMP showed 100% inclusivity and exclusivity, and a detection limit similar to that of multiplex PCR. In addition, we observed and corrected preferential amplification induced by what we call LAMP selection in the multiplex LAMP reaction. In conclusion, our assay was rapid, specific, and quantitative, making it a useful tool for the food industry. PMID:28349967

  3. Computation of ground motion amplification in Kolkata megacity (India) using finite-difference method for seismic microzonation

    NASA Astrophysics Data System (ADS)

    Shiuly, Amit; Kumar, Vinay; Narayan, Jay

    2014-06-01

    This paper presents the ground motion amplification scenario along with fundamental frequency (F 0) of sedimentary deposit for the seismic microzonation of Kolkata City, situated on the world's largest delta island with very soft soil deposit. A 4th order accurate SH-wave viscoelastic finite-difference algorithm is used for computation of response of 1D model for each borehole location. Different maps, such as for F 0, amplification at F 0, average spectral amplification (ASA) in the different frequency bandwidth of earthquake engineering interest are developed for a variety of end-users communities. The obtained ASA of the order of 3-6 at most of the borehole locations in a frequency range of 0.25-10.0 Hz reveals that Kolkata City may suffer severe damage even during a moderate earthquake. Further, unexpected severe damage to collapse of multi-storey buildings may occur in localities near Hoogly River and Salt Lake area due to double resonance effects during distant large earthquakes.

  4. 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.

  5. Method of increasing conversion of a fatty acid to its corresponding dicarboxylic acid

    DOEpatents

    Craft, David L.; Wilson, C. Ron; Eirich, Dudley; Zhang, Yeyan

    2004-09-14

    A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

  6. Method to detect the end-point for PCR DNA amplification using an ionically labeled probe and measuring impedance change

    DOEpatents

    Miles, Robin R.; Belgrader, Phillip; Fuller, Christopher D.

    2007-01-02

    Impedance measurements are used to detect the end-point for PCR DNA amplification. A pair of spaced electrodes are located on a surface of a microfluidic channel and an AC or DC voltage is applied across the electrodes to produce an electric field. An ionically labeled probe will attach to a complementary DNA segment, and a polymerase enzyme will release the ionic label. This causes the conductivity of the solution in the area of the electrode to change. This change in conductivity is measured as a change in the impedance been the two electrodes.

  7. 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

  8. Evaluation of rapid and sensitive reverse transcription loop-mediated isothermal amplification method for detecting infectious pancreatic necrosis virus in chum salmon (Oncorhynchus keta).

    PubMed

    Suebsing, Rungkarn; Oh, Myung-Joo; Kim, Jeong-Ho

    2011-07-01

    Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detecting Infectious pancreatic necrosis virus (IPNV) in chum salmon (Oncorhynchus keta) in Korea. The RT-LAMP is a novel approach of nucleic acid gene amplification with high specificity, sensitivity, and rapidity under isothermal conditions. Based on the VP2/NS gene sequence of VR-299 and Jasper strains, a set of 6 IPNV-specific primers was designed to recognize 8 diverse sequences of the IPNV RNA. The assay was successfully optimized to detect IPNV at 65°C in 30 min. The detection limit was 0.075 tissue culture infectious dose infecting 50% of inoculated cultures per milliliter (TCID(50)/ml) from IPNV-infected rainbow trout gonad (RTG)-2 cells, whereas nested reverse transcription polymerase chain reaction (nRT-PCR) had a sensitivity of 7.5 TCID(50)/ml. Using RT-LAMP assay, field samples were analyzed and the results compared with those of nRT-PCR assay. Two hundred and sixty-six out of 659 (40.4%) samples were IPNV-positive by RT-LAMP, whereas 182 of 659 samples (27.6%) were IPNV-positive by nRT-PCR. The results indicate that RT-LAMP can be a useful tool for early field diagnosis of IPNV.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  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. 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.

  16. Development and validation of a whole genome amplification long-range PCR sequencing method for ADPKD genotyping of low-level DNA samples.

    PubMed

    Liu, Genyan; Tan, Adrian Y; Michaeel, Alber; Blumenfeld, Jon; Donahue, Stephanie; Bobb, Warren; Parker, Tom; Levine, Daniel; Rennert, Hanna

    2014-10-15

    Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two large genes, PKD1 and PKD2, but genetic testing is complicated by the large transcript sizes and the duplication of PKD1 exons 1-33 as six pseudogenes on chromosome 16. Long-range PCR (LR-PCR) represents the gold standard approach for PKD1 genetic analysis. However, a major issue with this approach is that it requires large quantities of genomic DNA (gDNA) material limiting its application primarily to DNA extracted from blood. In this study, we have developed a whole genome amplification (WGA)-based genotyping assay for PKD1 and PKD2, and examined whether this approach can be applied to biosamples with low DNA yield, including blood, buccal cells and urine. DNA samples were amplified by multiple displacement amplification (MDA) and a high-fidelity DNA polymerase followed by LR-PCR and exon-specific amplifications of PKD1 and PKD2 respectively, and Sanger sequencing. This method has generated large amounts of DNA with high average product length (>10 kb), which were uniformly amplified across all sequences assessed. When compared to the gDNA direct sequencing method for six ADPKD samples, a total of 89 variants were detected including all 86 variations previously reported, in addition to three new variations, including one pathogenic mutation not previously detected by the standard gDNA-based analysis. We have further applied WGA to ADPKD mutation analysis of low DNA-yield specimens, successfully detecting all 63 gene variations. Compared to the gDNA method the WGA-based assay had a sensitivity and specificity of 100%. In conclusion, WGA-based LR-PCR represents a major technical improvement for PKD genotyping from trace amounts of DNA.

  17. 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.

  18. A novel method for diagnosis of smear-negative tuberculosis patients by combining a random unbiased Phi29 amplification with a specific real-time PCR.

    PubMed

    Pang, Yu; Lu, Jie; Yang, Jian; Wang, Yufeng; Cohen, Chad; Ni, Xin; Zhao, Yanlin

    2015-07-01

    In this study, we develop a novel method for diagnosis of smear-negative tuberculosis patients by performing a random unbiased Phi29 amplification prior to the use of a specific real-time PCR. The limit of detection (LOD) of the conventional real-time PCR was 100 colony-forming units (CFU) of MTB genome/reaction, while the REPLI real-time PCR assay could detect 0.4 CFU/reaction. In comparison with the conventional real-time PCR, REPLI real-time PCR shows better sensitivity for the detection of smear-negative tuberculosis (P = 0.015).

  19. Loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia in HIV-uninfected immunocompromised patients with pulmonary infiltrates.

    PubMed

    Nakashima, Kei; Aoshima, Masahiro; Ohkuni, Yoshihiro; Hoshino, Eri; Hashimoto, Kohei; Otsuka, Yoshihito

    2014-12-01

    Loop-mediated isothermal amplification (LAMP) is becoming an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We retrospectively evaluated 78 consecutive HIV-uninfected patients who underwent LAMP method for diagnosing Pneumocystis pneumonia (PCP). Diagnosis of PCP was made by the detection of Pneumocystis jirovecii (P. jirovecii) with positive LAMP or conventional staining (CS) (Grocott methenamine silver staining or Diff-Quick™) on the basis of compatible clinical symptoms and radiologic findings. Additionally, we reviewed HIV-uninfected immunocompromised patients who underwent subcontract PCR as a historical control. LAMP was positive in 10 (90.9%) of 11 positive-CS patients. Among 13 negative-CS patients with positive LAMP, 11 (84.6%) had PCP, and the remaining 2 were categorized as having P. jirovecii colonization. LDH levels in negative-CS PCP were higher than in positive-CS PCP (p = 0.026). (1 → 3)-β-D-glucan levels in negative-CS PCP were lower than in positive-CS PCP (p = 0.011). The interval from symptom onset to diagnosis as PCP in LAMP group (3.45 ± 1.77 days; n = 22) was shorter than in subcontract PCR group (6.90 ± 2.28 days; n = 10; p < 0.001). As for patients without PCP, duration of unnecessary PCP treatment in LAMP group (2; 2-3 days; n = 10) was shorter than in subcontract PCR group (7; 7-12.25 days; n = 6; p = 0.003). LAMP showed higher sensitivity (95.4%) and positive predictive value (91.3%) than subcontract PCR did. Pneumocystis LAMP method is a sensitive and cost-effective diagnostic method and is easy to administer in general hospitals. In-house LAMP method would realize early diagnosis of PCP, resulting in improving PCP prognosis and reducing unnecessary PCP-specific treatment.

  20. 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.

  1. 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.

  2. Precise characterization method of antibody-conjugated magnetic nanoparticles for pathogen detection using stuffer-free multiplex ligation-dependent probe amplification.

    PubMed

    Chung, Boram; Shin, Gi Won; Choi, Woong; Joo, Jinmyoung; Jeon, Sangmin; Jung, Gyoo Yeol

    2014-12-01

    Antibody-conjugated magnetic nanoparticles (Ab-MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab-MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab-MNPs as a pathogen detection method requires accurate characterization of Ab-MNP capture ability and standardization of all handling processes. In this study, we used high-resolution CE-single strand conformational polymorphism coupled with a stuffer-free multiplex ligation-dependent probe amplification system to characterize Ab-MNPs. The capture ability of Ab-MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab-MNPs was also assessed. The results showed that the stuffer-free multiplex ligation-dependent probe amplification system has the potential to serve as a standard characterization method for Ab-MNPs. Moreover, the precise characterization of Ab-MNPs facilitated robust pathogen detection in various applications.

  3. A loop mediated isothermal amplification (LAMP) method for detection of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Gunimaladevi, I; Kono, T; Lapatra, S E; Sakai, M

    2005-05-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol was developed for detection of infectious hematopoietic necrosis virus (IHNV) RNA in rainbow trout (Oncorhynchus mykiss). A set of four primers, two outer and two inner primers for the RT-LAMP and the LAMP assay, were designed based on the sequence of G-protein of IHNV. Time and temperature conditions were optimized for 60 min at 63 degrees C for both RT-LAMP and LAMP protocols. The detection limit was found to be similar for both RT-LAMP and LAMP. When the sensitivity of RT-LAMP and LAMP were compared with conventional nested PCR, a10-fold higher sensitivity was seen for the LAMP protocols.

  4. 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.

  5. An online field-amplification sample stacking method for the determination of diuretics in urine by capillary electrophoresis-amperometric detection.

    PubMed

    Zheng, Xinyu; Lu, Minghua; Zhang, Lan; Chi, Yuwu; Zheng, Lihui; Chen, Guonan

    2008-06-30

    A simple and sensitive online field-amplification sample stacking (FASS) pre-enrichment method following by capillary electrophoresis with amperometric detection has been developed for the determination of diuretics, such as indapamide (IDP), hydrochlorothiazide (HCT) and bumetanide (BMTN) in urine. Under the optimum conditions, it was found that the low concentration buffer solution could be used as the diluents for simultaneous field-amplification injection of three diuretics after electrokinetically injecting a short water plug (15 kV, 3 s). Three analytes could be well separated within 10 min in an uncoated fused-silica capillary with H(3)BO(3)-Na(2)B(4)O(7) (BB) buffer solution (pH 8.98). The detection limits (S/N=3) were 9.0 ng/mL for IDP, 20 ng/mL for HCT and 1.5 ng/mL for BMTN, respectively. The detection limits of three diuretics were much lower by FASS than that by conventional sample injection, of which the detection limits were 340, 890 and 330 ng/mL for IDP, HCT and BMTN, respectively. Especially, for bumetanide the detection limit was 220-time lower by FASS. The linear ranges of three diuretics were all over three orders of magnitude. The proposed method has been successfully applied to analyze the diuretics in human urine samples without off-column sample pre-concentration.

  6. Fast and simple detection methods for the 4-base pair deletion of canine MDR1/ ABCB1 gene by PCR and isothermal amplification.

    PubMed

    Stiedl, Cathrin P; Weber, Karin

    2017-03-01

    Dogs with a 4-bp deletion in the MDR1 (or ABCB1) gene show intolerance to certain drugs routinely used in veterinary medicine, such as ivermectin, vincristine, and doxorubicin. The mutation leads to a dysfunctional P-glycoprotein drug transporter, which results in drug accumulation in the brain and severe neurotoxicity. A rapid and accurate in-house test to determine the genotype of patients in cases of acute neurotoxic signs or in tumor patients is desirable. We describe a cost-effective detection method with simple technical equipment for veterinary practice. Two allele-specific methods are presented, which allow discrimination of all genotypes, require little hands-on time, and show the results within ~1 h after DNA sampling. DNA from buccal swabs of 115 dogs with known genotype (no mutation, n = 54; heterozygous for the mutation, n = 37; homozygous for the mutation, n = 24) was extracted either by using a column-based extraction kit or by heating swabs in a simple NaOH-Tris buffer. Amplification was performed either by allele-specific fast polymerase chain reaction or by allele-specific loop-mediated isothermal amplification (LAMP). Analysis was done either on agarose gels, by simple endpoint visualization using ultraviolet light, or by measuring the increase of fluorescence and time to threshold crossing. Commercial master mixes reduced the preparation time and minimized sources of error in both methods. Both methods allowed the discrimination of all 3 genotypes, and the results of the new methods matched the results of the previous genotyping. The presented methods could be used for fast individual MDR1/ ABCB1 genotyping with less equipment than existing methods.

  7. Comparison of extraction methods for the recovery, amplification and species-specific analysis of DNA from bone and bone meals.

    PubMed

    Prado, Marta; Franco, Carlos M; Fente, Cristina A; Cepeda, Alberto; Vázquez, Beatriz I; Barros-Velázquez, Jorge

    2002-04-01

    We report the effect of several parameters on the efficiency of recovery of DNA from animal bones. The effects of preheating the samples (at either 60 degrees C or 100 degrees C) at different intervals (from 1 h to overnight) in different media (water, 0.5 M ethylenediaminetetraacetic acid (EDTA), or 0.5 M EDTA + 0.05% sodium dodecyl sulfate (SDS) were investigated. The effect of slight (5 min) or intense (30 min) pretreatments with ultrasound was also evaluated. Several different treatments with proteinase K (ranging from 200 to 800 microg, and lasting from 1 to 3 h) at 65 degrees C were also considered. Additionally, two different DNA extraction methods (based on silica resins and purification columns, respectively) were evaluated. The recovery of DNA from the samples was 40% higher when the bones were preheated in 0.5 M EDTA at 60 degrees C for 1 h, this being followed by treatment with 800 microg of proteinase K for 3 h. The DNA thus obtained was successfully amplified by polymerase chain reaction (PCR) using a set of primers specific to a 359 bp region of the mitochondrial cytochrome b gene, and the species of origin were identified by visualizing the restriction fragment length polymorphism (RFLP) with the endonucleases PalI and MboI.

  8. Characterization and Multicentric Validation of a Common Standard for Toxoplasma gondii Detection Using Nucleic Acid Amplification Assays

    PubMed Central

    Varlet-Marie, Emmanuelle; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Filisetti, Denis; Pelloux, Hervé; Touafek, Fériel; Yera, Hélène

    2014-01-01

    The molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and suffers from lack of standardization, hence the large diversity of performances between laboratories. Moreover, quantifications of parasitic loads differ among centers, a fact which prevents the possible prediction of the severity of this disease as a function of parasitic loads. The objectives of this multicentric study performed in eight proficient laboratories of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (NRC-T) were (i) to assess the suitability of a lyophilized preparation of Toxoplasma gondii as a common standard for use in this PCR-based molecular diagnosis and (ii) to make this standard available to the community. High-quality written procedures were used for the production and qualification of this standard. Three independent batches of this standard, containing concentrations ranging from 104 to 0.01 T. gondii genome equivalents per PCR, were first assessed: the linear dynamic range was ≥6 log, the intra-assay coefficients of variation (CV) from a sample containing 10 T. gondii organisms per PCR were 0.3% to 0.42%, and the interassay CV over a 2-week period was 0.76% to 1.47%. A further assessment in eight diagnostic centers showed that the standard is stable, robust, and reliable. These lyophilized standards can easily be produced at a larger scale when needed and can be made widely available at the national level. To our knowledge, this is the first quality control assessment of a common standard which is usable both for self-evaluation in laboratories and for accurate quantification of parasitic loads in T. gondii prenatal infections. PMID:25187637

  9. Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays.

    PubMed

    Varlet-Marie, Emmanuelle; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Filisetti, Denis; Pelloux, Hervé; Touafek, Fériel; Yera, Hélène; Bastien, Patrick

    2014-11-01

    The molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and suffers from lack of standardization, hence the large diversity of performances between laboratories. Moreover, quantifications of parasitic loads differ among centers, a fact which prevents the possible prediction of the severity of this disease as a function of parasitic loads. The objectives of this multicentric study performed in eight proficient laboratories of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (NRC-T) were (i) to assess the suitability of a lyophilized preparation of Toxoplasma gondii as a common standard for use in this PCR-based molecular diagnosis and (ii) to make this standard available to the community. High-quality written procedures were used for the production and qualification of this standard. Three independent batches of this standard, containing concentrations ranging from 10(4) to 0.01 T. gondii genome equivalents per PCR, were first assessed: the linear dynamic range was ≥ 6 log, the intra-assay coefficients of variation (CV) from a sample containing 10 T. gondii organisms per PCR were 0.3% to 0.42%, and the interassay CV over a 2-week period was 0.76% to 1.47%. A further assessment in eight diagnostic centers showed that the standard is stable, robust, and reliable. These lyophilized standards can easily be produced at a larger scale when needed and can be made widely available at the national level. To our knowledge, this is the first quality control assessment of a common standard which is usable both for self-evaluation in laboratories and for accurate quantification of parasitic loads in T. gondii prenatal infections.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

    PubMed

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings.

  16. Development and application of a reverse transcription loop-mediated isothermal amplification method for rapid detection of Duck hepatitis A virus type 1.

    PubMed

    Yang, Limin; Li, Jing; Bi, Yuhai; Xu, Lei; Liu, Wenjun

    2012-12-01

    We developed and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting Duck hepatitis A virus type 1 (DHAV-1). The amplification could be finished in 1 h under isothermal conditions at 63 °C by employing a set of four primers targeting the 2C gene of DHAV-1. The RT-LAMP assay showed higher sensitivity than the RT-PCR with a detection limit of 0.1 ELD(50) 0.1 ml(-1) of DHAV-1. The RT-LAMP assay was highly specific; no cross-reactivity was observed from the samples of other related viruses, bacteria, allantoic fluid of normal chicken embryos, or the livers of uninfected ducks. Thirty clinical samples were subjected to detection by RT-LAMP, RT-PCR, and virus isolation, which obtained completely consistent, positive results. As a simple, rapid, and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of DHAV-1.

  17. Development of a Rapid, Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

    PubMed Central

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings. PMID:25822175

  18. Rapid detection of Bombyx mori nucleopolyhedrovirus (BmNPV) by loop-mediated isothermal amplification assay combined with a lateral flow dipstick method.

    PubMed

    Zhou, Yang; Wu, Jiege; Lin, Feng; Chen, Naifu; Yuan, Shaofei; Ding, Lina; Gao, Li; Hang, Bangxing

    2015-12-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the domestic silkworm. The disease often breaks out in sericultural countries and due to its high infectivity; it is difficult to control, resulting in heavy economic loss. In order to develop a rapid, sensitive visual detection and simple-to-use novel technology for detection of BmNPV, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) method was described. In this study, a set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the BmNPV gp41 gene, and the LAMP for the detection of BmNPV was developed by isothermal amplification at 61 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min. The detection limit of LAMP-LFD was 0.2 pg DNA extracted from silkworm infected with BmNPV and was 100 times more sensitive than conventional PCR. No product was generated from silkworm infected with other viruses. Furthermore, we applied the technique to detect BmNPV in the hemolymph and feces at different intervals post infection (pi). In conclusion, the novel LAMP-LFD setup presented here is simple, rapid, reliable, and has the potential for future use in the detection of BmNPV.

  19. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field.

  20. 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.

  1. Molecular epidemiology and a loop-mediated isothermal amplification method for diagnosis of infection with rabies virus in Zambia.

    PubMed

    Muleya, Walter; Namangala, Boniface; Mweene, Aaron; Zulu, Luke; Fandamu, Paul; Banda, Douglas; Kimura, Takashi; Sawa, Hirofumi; Ishii, Akihiro

    2012-01-01

    The National Livestock Epidemiology and Information Center (NALEIC) in Zambia reported over 132 cases of canine rabies diagnosed by the direct fluorescent antibody test (DFAT) from 2004 to 2009. In this study, the lineage of rabies virus (RABV) in Zambia was determined by phylogenetic analysis of the nucleoprotein (N) and glycoprotein (G) gene sequences. Total RNA was extracted from 87-DFAT brain specimens out of which only 35 (40%) were positive on nested reverse transcription polymerase chain reaction (RT-PCR) for each gene, and 26 being positive for both genes. Positive specimens for the N (n=33) and G (n=35) genes were used for phylogenetic analysis. Phylogenetic analysis of the N gene showed two phylogenetic clusters in Zambia belonging to the Africa 1b lineage present in eastern and southern Africa. While one cluster exclusively comprised Zambian strains, the other was more heterogeneous regarding the RABV origins and included strains from Tanzania, Mozambique and Zambia. Phylogenetic analysis of the G gene revealed similar RABV strains in different hosts and regions of Zambia. We designed primers for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from the consensus sequence of the N gene in an attempt to improve the molecular diagnosis of RABV in Zambia. The specificity and reproducibility of the RT-LAMP assay was confirmed with actual clinical specimens. Therefore, the RT-LAMP assay presented in this study may prove to be useful for routine diagnosis of rabies in Zambia.

  2. Method for nucleic acid isolation using supercritical fluids

    SciTech Connect

    Nivens, David E.; Applegate, Bruce M.

    1999-01-01

    A method for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification.

  3. Method for nucleic acid isolation using supercritical fluids

    DOEpatents

    Nivens, D.E.; Applegate, B.M.

    1999-07-13

    A method is disclosed for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification. 4 figs.

  4. Instant, Visual, and Instrument-Free Method for On-Site Screening of GTS 40-3-2 Soybean Based on Body-Heat Triggered Recombinase Polymerase Amplification.

    PubMed

    Wang, Rui; Zhang, Fang; Wang, Liu; Qian, Wenjuan; Qian, Cheng; Wu, Jian; Ying, Yibin

    2017-04-06

    On-site monitoring the plantation of genetically modified (GM) crops is of critical importance in agriculture industry throughout the world. In this paper, a simple, visual and instrument-free method for instant on-site detection of GTS 40-3-2 soybean has been developed. It is based on body-heat recombinase polymerase amplification (RPA) and followed with naked-eye detection via fluorescent DNA dye. Combining with extremely simplified sample preparation, the whole detection process can be accomplished within 10 min and the fluorescent results can be photographed by an accompanied smart phone. Results demonstrated a 100% detection rate for screening of practical GTS 40-3-2 soybean samples by 20 volunteers under different ambient temperatures. This method is not only suitable for on-site detection of GM crops but also demonstrates great potential to be applied in other fields.

  5. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1998-07-21

    A method is disclosed for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe. 11 figs.

  6. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1998-01-01

    A method for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe.

  7. Continuous-flow free acid monitoring method and system

    DOEpatents

    Strain, James E.; Ross, Harley H.

    1981-01-01

    A free acid monitoring method and apparatus is provided for continuously measuring the excess acid present in a process stream. The disclosed monitoring system and method is based on the relationship of the partial pressure ratio of water and acid in equilibrium with an acid solution at constant temperature. A portion of the process stream is pumped into and flows through the monitor under the influence of gravity and back to the process stream. A continuous flowing sample is vaporized at a constant temperature and the vapor is subsequently condensed. Conductivity measurements of the condensate produces a nonlinear response function from which the free acid molarity of the sample process stream is determined.

  8. Continuous-flow free acid monitoring method and system

    DOEpatents

    Strain, J.E.; Ross, H.H.

    1980-01-11

    A free acid monitoring method and apparatus is provided for continuously measuring the excess acid present in a process stream. The disclosed monitoring system and method is based on the relationship of the partial pressure ratio of water and acid in equilibrium with an acid solution at constant temperature. A portion of the process stream is pumped into and flows through the monitor under the influence of gravity and back to the process stream. A continuous flowing sample is vaporized at a constant temperature and the vapor is subsequently condensed. Conductivity measurements of the condensate produces a nonlinear response function from which the free acid molarity of the sample process stream is determined.

  9. Sulfuric acid thermoelectrochemical system and method

    DOEpatents

    Ludwig, Frank A.

    1989-01-01

    A thermoelectrochemical system in which an electrical current is generated between a cathode immersed in a concentrated sulfuric acid solution and an anode immersed in an aqueous buffer solution of sodium bisulfate and sodium sulfate. Reactants consumed at the electrodes during the electrochemical reaction are thermochemically regenerated and recycled to the electrodes to provide continuous operation of the system.

  10. Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

    SciTech Connect

    Cary, Robert E.

    2015-12-08

    Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

  11. Methods of refining and producing isomerized fatty acid esters and fatty acids from natural oil feedstocks

    DOEpatents

    Snead, Thomas E.; Cohen, Steven A.; Gildon, Demond L.; Beltran, Leslie V.; Kunz, Linda A.; Pals, Tessa M.; Quinn, Jordan R; Behrends, Jr., Raymond T.; Bernhardt, Randal J.

    2016-07-05

    Methods are provided for refining natural oil feedstocks and producing isomerized esters and acids. The methods comprise providing a C4-C18 unsaturated fatty ester or acid, and isomerizing the fatty acid ester or acid in the presence of heat or an isomerization catalyst to form an isomerized fatty ester or acid. In some embodiments, the methods comprise forming a dibasic ester or dibasic acid prior to the isomerizing step. In certain embodiments, the methods further comprise hydrolyzing the dibasic ester to form a dibasic acid. In certain embodiments, the olefin is formed by reacting the feedstock in the presence of a metathesis catalyst under conditions sufficient to form a metathesized product comprising olefins and esters, separating the olefins from the esters in the metathesized product, and transesterifying the esters in the presence of an alcohol to form a transesterified product having unsaturated esters.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Boron containing amino acid compounds and methods for their use

    DOEpatents

    Glass, John D.; Coderre, Jeffrey A.

    2000-01-01

    The present invention provides new boron containing amino acid compounds and methods for making these compounds by contacting melphalan or another nitrogen mustard derivative and sodium borocaptate. The present invention also provides a method of treating a mammal having a tumor by administering to the mammal a therapeutically effective amount of the new boron containing amino acid compounds.

  17. Boron containing amino acid compounds and methods for their use

    SciTech Connect

    Glass, J.D.; Coderre, J.A.

    2000-01-25

    The present invention provides new boron containing amino acid compounds and methods for making these compounds by contacting melphalan or another nitrogen mustard derivative and sodium borocaptate. The present invention also provides a method of treating a mammal having a tumor by administering to the mammal a therapeutically effective amount of the new boron containing amino acid compounds.

  18. A novel and rapid diagnostic method for discriminating between feces of sika deer and Japanese serow by loop-mediated isothermal amplification.

    PubMed

    Aikawa, T; Horino, S; Ichihara, Y

    2015-08-01

    Severe damages to natural vegetation, agriculture, and forestry caused by overpopulation of sika deer (Cervus nippon) have markedly increased in Japan in recent years. To devise a population management plan of sika deer, information on the distribution and population size of the animal in each region is indispensable. An easy and effective method to obtain this information is to count the fecal pellets in the field. However, the habitat of sika deer in Japan overlaps that of Japanese serow (Capricornis crispus). Additionally, it is difficult to discriminate between the feces of both animals. Here, we present a rapid and precise diagnostic method for discriminating between the feces of sika deer and Japanese serow using loop-mediated isothermal amplification (LAMP) targeting cytochrome b gene in the mitochondrial DNA. Our results showed that the LAMP can discriminate between the feces of sika deer and Japanese serow, and the method is simpler and more sensitive than the conventional molecular diagnostic method. Since LAMP method does not require special skills for molecular biology techniques, even the field researchers who have never done a molecular experiment can easily carry out the protocol. In addition, the entire protocol, from DNA extraction from fecal pellet to identification of species, takes only about 75 min and does not require expensive equipment. Hence, this diagnostic method is simple, fast, and accessible to anyone. As such, the method can be a useful tool to estimate distribution and population size of sika deer.

  19. 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

  20. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John; Whittle, Edward J.

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  1. Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms.

    PubMed

    Suebsing, Rungkarn; Kampeera, Jantana; Sirithammajak, Sarawut; Withyachumnarnkul, Boonsirm; Turner, Warren; Kiatpathomchai, Wansika

    2015-03-01

    Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP-calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10(2) F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP-calcein method had 97% homology with the DNA sequence of F. columnare.

  2. Evaluation of a novel method based on amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting) for typing strains of vancomycin-resistant Enterococcus faecium.

    PubMed

    Krawczyk, Beata; Lewandowski, Krzysztof; Bronk, Marek; Samet, Alfred; Myjak, Przemysław; Kur, Józef

    2003-03-01

    In the search for an effective DNA-typing technique for use in hospital epidemiology, the performance and convenience of a novel assay based on the fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting) was tested. A large number of vancomycin-resistant Enterococcus faecium (VREM) isolates from haematological ward patients of the Clinical Hospital in Gdańsk were examined. We found that ADSRRS fingerprinting analysis is a rapid method that offers good discriminatory power. The method demonstrated also excellent reproducibility. The usefulness of the ADSRRS fingerprinting method for molecular typing was compared with pulsed field gel electrophoresis (PFGE) method, which is currently considered the gold standard for molecular typing of isolates recovered from patients and the environment in the course of investigation and control of nosocomial outbreaks. Clustering of ADSRRS fingerprinting data matched pulsed field gel electrophoresis data. The features of ADSRRS fingerprinting technique is discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning VREM that may occur in a single medical ward.

  3. Evaluation of a Loop-Mediated Isothermal Amplification Method as a Tool for Diagnosis of Infection by the Zoonotic Simian Malaria Parasite Plasmodium knowlesi▿

    PubMed Central

    Iseki, Hiroshi; Kawai, Satoru; Takahashi, Nobuyuki; Hirai, Makoto; Tanabe, Kazuyuki; Yokoyama, Naoaki; Igarashi, Ikuo

    2010-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel method that rapidly amplifies target DNA with high specificity under isothermal conditions. It has been applied as a diagnostic tool for several infectious diseases, including viral, bacterial, and parasitic diseases. In the present study, we developed a LAMP method for the molecular diagnosis of Plasmodium knowlesi infection (PkLAMP) and evaluated its sensitivity, specificity, and clinical applicability. We designed three sets of PkLAMP primers for the species-specific β-tubulin gene. The primer sets for PkLAMP specifically amplified the autologous DNA extracts of P. knowlesi, and the sensitivity of the test was 100-fold that of single-PCR assay. These results indicate that our PkLAMP method can be used to efficiently distinguish between P. knowlesi and other malaria parasites. To evaluate the feasibility of using in vivo materials, comparisons of PkLAMP and the conventional nested PCR (nPCR) method and microscopic examination were made with blood samples from two experimentally infected monkeys. These studies showed that P. knowlesi infection can be identified much earlier with PkLAMP than with nPCR and microscopy. Moreover, the detection performance of PkLAMP using whole blood as the template was identical to that of PkLAMP when genomic DNA extracts were used. These results suggest that the PkLAMP method is a promising tool for molecular diagnosis of P. knowlesi infection in areas of endemicity. PMID:20444968

  4. Evaluation of a loop-mediated isothermal amplification method as a tool for diagnosis of infection by the zoonotic simian malaria parasite Plasmodium knowlesi.

    PubMed

    Iseki, Hiroshi; Kawai, Satoru; Takahashi, Nobuyuki; Hirai, Makoto; Tanabe, Kazuyuki; Yokoyama, Naoaki; Igarashi, Ikuo

    2010-07-01

    Loop-mediated isothermal amplification (LAMP) is a novel method that rapidly amplifies target DNA with high specificity under isothermal conditions. It has been applied as a diagnostic tool for several infectious diseases, including viral, bacterial, and parasitic diseases. In the present study, we developed a LAMP method for the molecular diagnosis of Plasmodium knowlesi infection (PkLAMP) and evaluated its sensitivity, specificity, and clinical applicability. We designed three sets of PkLAMP primers for the species-specific beta-tubulin gene. The primer sets for PkLAMP specifically amplified the autologous DNA extracts of P. knowlesi, and the sensitivity of the test was 100-fold that of single-PCR assay. These results indicate that our PkLAMP method can be used to efficiently distinguish between P. knowlesi and other malaria parasites. To evaluate the feasibility of using in vivo materials, comparisons of PkLAMP and the conventional nested PCR (nPCR) method and microscopic examination were made with blood samples from two experimentally infected monkeys. These studies showed that P. knowlesi infection can be identified much earlier with PkLAMP than with nPCR and microscopy. Moreover, the detection performance of PkLAMP using whole blood as the template was identical to that of PkLAMP when genomic DNA extracts were used. These results suggest that the PkLAMP method is a promising tool for molecular diagnosis of P. knowlesi infection in areas of endemicity.

  5. Compositions and method for controlling precipitation when acidizing sour wells

    SciTech Connect

    Dill, W.R.; Walker, M.L.

    1990-08-21

    This patent describes a method of treating a sour well penetrating a subterranean formation. It comprises: introducing into the well a treating fluid comprising an acid solution having a pH below 1.9, an iron sequestering agent comprising at least one compound selected from the group consisting of aminopolycarboxylic acids, hydroxycarboxylic acids, cyclic polyethers and derivatives of the acids and ethers, present in an amount of from about 0.25 to about 5 percent by weight of the acid solution, and a sulfide modifier comprising at least one compound selected from the group consisting of an aldehyde, acetal, hemiacetal and any other compound capable of forming aldehydes in the acid solution, present in an amount of from about 0.25 to about 5 percent of the acid solution; and treating the subterranean formation with the treating fluid.

  6. 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.

  7. Fluorometric Method for Determination of Uric Acid in Flour

    DTIC Science & Technology

    1980-07-01

    of the excreta of adult Tribolium used in this study for natural infestation, accumulates in infested products providing a quantitative measure of past...by Adult Tribolium 8 4 FLUOROMETRIC METHOD FOR DETERMINATION OF URIC ACID IN FLOUR Introduction Chemical and microanalytical techniques have been...determining uric acid content.’ Approximately 18% of the total excreta from Tribolium confusum is uric acid.2 Various investigators have established

  8. Development of a method to recovery and amplification DNA by real-time PCR from commercial vegetable oils.

    PubMed

    Ramos-Gómez, Sonia; Busto, María D; Perez-Mateos, Manuel; Ortega, Natividad

    2014-09-01

    This study describes the design of a suitable DNA isolation method from commercial vegetable oils for the application of DNA markers for food safety and traceability. Firstly, a comparative study was made of eight methods for the recovery of high quality DNA from olive, sunflower and palm oils, and a CTAB-based method was selected. In order to optimize this method, the effect of the organic compounds and several components in the lysis buffer and the lysis and precipitation time were evaluated. For the purpose of overcoming the limitations detected in spectrophotometric and PCR DNA yield evaluations, the performance of the extraction protocols during the optimization processes was evaluated using qPCR. The suggested DNA extraction optimized is less time consuming than other conventional DNA extraction methods, uses a reduced oil volume and is cheaper than available commercial kits. Additionally, the applicability of this method has been successfully assayed in ten commercial vegetable oils and derivatives.

  9. Human jagged polypeptide, encoding nucleic acids and methods of use

    DOEpatents

    Li, Linheng; Hood, Leroy

    2000-01-01

    The present invention provides an isolated polypeptide exhibiting substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the polypeptide does not have the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. The invention further provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the nucleotide sequence does not encode the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Also provided herein is a method of inhibiting differentiation of hematopoietic progenitor cells by contacting the progenitor cells with an isolated JAGGED polypeptide, or active fragment thereof. The invention additionally provides a method of diagnosing Alagille Syndrome in an individual. The method consists of detecting an Alagille Syndrome disease-associated mutation linked to a JAGGED locus.

  10. Method for incorporating radioactive phosphoric acid solutions in concrete

    DOEpatents

    Wolf, G.A.; Smith, J.W.; Ihle, N.C.

    1982-07-08

    A method for incorporating radioactive phosphoric acid solutions in concrete is described wherein the phosphoric acid is reacted with Ca(OH)/sub 2/ to form a precipitate of hydroxyapatite and the hydroxyapatite is mixed with Portland cement to form concrete.

  11. Method for incorporating radioactive phosphoric acid solutions in concrete

    DOEpatents

    Wolf, Gary A [Kennewick, WA; Smith, Jeffrey W [Lancaster, OH; Ihle, Nathan C [Walla Walla, WA

    1984-01-01

    A method for incorporating radioactive phosphoric acid solutions in concrete is described wherein the phosphoric acid is reacted with Ca(OH).sub.2 to form a precipitate of hydroxyapatite and the hydroxyapatite is mixed with portland cement to form concrete.

  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. A novel gene trap method using terminator-REMI and 3' rapid amplification of cDNA ends (RACE) in Dictyostelium.

    PubMed

    Takeda, Kosuke; Saito, Tamao; Tanaka, Tomoyuki; Morio, Takahiro; Maeda, Mineko; Tanaka, Yoshimasa; Ochiai, Hiroshi

    2003-07-17

    We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3' proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3' rapid amplification of cDNA ends (3'-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3' ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3' sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants.

  14. Quantitative real-time polymerase chain reaction is an alternative method for the detection of HER-2 amplification in formalin-fixed paraffin-embedded breast cancer samples.

    PubMed

    Pu, Tianjie; Guo, Peng; Qiu, Yan; Chen, Shinan; Yang, Libo; Sun, Linyong; Ye, Feng; Bu, Hong

    2015-01-01

    Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are the most common methods that are used to quantify HER-2 gene and protein levels, respectively, in human breast cancer. However, due to bad sample quality, some samples are unable to be subjected to a FISH assay. We evaluated 71 formalin-fixed paraffin-embedded (FFPE) breast carcinoma specimens by quantitative real-time polymerase chain reaction (qPCR), IHC, and FISH. We also performed qPCR and FISH assays on delayed formalin-fixed (DDF) samples. The qPCR results were in complete concordance with the results of IHC and FISH. In regards to the DDF samples, the HER-2 fluorescent signal seemed decayed compared with that of the DDF samples after 1 h. However, the qPCR method still works well up to 12 hours. Our results indicated that qPCR was obviously superior to FISH in cases that were not fixed in a reasonable amount of time. However, qPCR can be an alternative method by which to perform HER2 amplification assays in breast cancer.

  15. Development of a Polymerase Chain Reaction (PCR) method based on amplification of mitochondrial DNA to detect Plasmodium falciparum and Plasmodium vivax.

    PubMed

    Cunha, Maristela G; Medina, Tiago S; Oliveira, Salma G; Marinho, Anderson N; Póvoa, Marinete M; Ribeiro-dos-Santos, Andrea K C

    2009-07-01

    In this study we standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) is amplified using a simple but sensitive PCR method as a tool to detect Plasmodium falciparum and Plasmodium vivax. Specific primers were designed to hybridize with cytochrome c oxidase genes of P. falciparum (cox III) and P. vivax (cox I). Amplification products were obtained for all positive samples, presenting homology only for species-specific mtDNA. Sensitivity and specificity were 100%. The applicability of the method was tested in a cross-sectional study, in which 88 blood samples from individuals naturally exposed to malaria in the Brazilian Amazon region were analyzed. Based on the results, the sensitivity and specificity were 100% and 88.3%, respectively. This simple and sensitive PCR method can be useful in specific situations and in different settings of malaria management, in endemic as well as non-endemic areas (travelers), and in clinical or epidemiological studies, with applications in malaria control programs.

  16. Simple differentiation method of mumps Hoshino vaccine strain from wild strains by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Yoshida, Naoko; Fujino, Motoko; Ota, Yoshinori; Notomi, Tsugunori; Nakayama, Tetsuo

    2007-01-26

    Mumps virus is still circulating and annual mumps outbreaks occur with fluctuating magnitudes in Japan. Aseptic meningitis has been reported after vaccination and it would be of importance to determine whether this was related to the vaccination. The objective of this study was to develop a sensitive, specific and rapid diagnostic method for the differentiation of the Hoshino vaccine strain from circulating wild types. We developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method of the hemagglutinin neuraminidase (HN) region for the detection of mumps virus genome from clinical samples. The typical ladder pattern disappeared after the LAMP products of the Hoshino vaccine strain were digested with ScaI, but those of wild types were not cut by ScaI. We obtained 19 cerebro spinal fluids (CSF) from the patients with aseptic meningitis and 17 salivary swab samples from the patients with acute parotitis after mumps vaccination, in which one case was complicated with orchitis. Mumps virus genome was detected in 18 CSF samples and in all NPS by RT-LAMP. The Hoshino vaccine strain was identified in 16 out of 18 CSF RT-LAMP positives and in 11 out of 17 NPS samples and the remaining samples were identified as wild types. RT-LAMP followed by ScaI digestion is a sensitive, simple and rapid differential method and useful for laboratory surveillance for vaccine-adverse events.

  17. 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

  18. 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.

  19. Methods and compositions for efficient nucleic acid sequencing

    DOEpatents

    Drmanac, Radoje

    2006-07-04

    Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

  20. Methods and compositions for efficient nucleic acid sequencing

    DOEpatents

    Drmanac, Radoje

    2002-01-01

    Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

  1. Improvement of the quantitation method for the tdh+ Vibrio parahaemolyticus in molluscan shellfish based on most-probable- number, immunomagnetic separation, and loop-mediated isothermal amplification

    PubMed Central

    Escalante-Maldonado, Oscar; Kayali, Ahmad Y.; Yamazaki, Wataru; Vuddhakul, Varaporn; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki

    2015-01-01

    Vibrio parahaemolyticus is a marine microorganism that can cause seafood-borne gastroenteritis in humans. The infection can be spread and has become a pandemic through the international trade of contaminated seafood. Strains carrying the tdh gene encoding the thermostable direct hemolysin (TDH) and/or the trh gene encoding the TDH-related hemolysin (TRH) are considered to be pathogenic with the former gene being the most frequently found in clinical strains. However, their distribution frequency in environmental isolates is below 1%. Thus, very sensitive methods are required for detection and quantitation of tdh+ strains in seafood. We previously reported a method to detect and quantify tdh+ V. parahaemolyticus in seafood. This method consists of three components: the most-probable-number (MPN), the immunomagnetic separation (IMS) targeting all established K antigens, and the loop-mediated isothermal amplification (LAMP) targeting the tdh gene. However, this method faces regional issues in tropical zones of the world. Technicians have difficulties in securing dependable reagents in high-temperature climates where we found MPN underestimation in samples having tdh+ strains as well as other microorganisms present at high concentrations. In the present study, we solved the underestimation problem associated with the salt polymyxin broth enrichment for the MPN component and with the immunomagnetic bead-target association for the IMS component. We also improved the supply and maintenance of the dependable reagents by introducing a dried reagent system to the LAMP component. The modified method is specific, sensitive, quick and easy and applicable regardless of the concentrations of tdh+ V. parahaemolyticus. Therefore, we conclude this modified method is useful in world tropical, sub-tropical, and temperate zones. PMID:25914681

  2. Improvement of the quantitation method for the tdh (+) Vibrio parahaemolyticus in molluscan shellfish based on most-probable- number, immunomagnetic separation, and loop-mediated isothermal amplification.

    PubMed

    Escalante-Maldonado, Oscar; Kayali, Ahmad Y; Yamazaki, Wataru; Vuddhakul, Varaporn; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki

    2015-01-01

    Vibrio parahaemolyticus is a marine microorganism that can cause seafood-borne gastroenteritis in humans. The infection can be spread and has become a pandemic through the international trade of contaminated seafood. Strains carrying the tdh gene encoding the thermostable direct hemolysin (TDH) and/or the trh gene encoding the TDH-related hemolysin (TRH) are considered to be pathogenic with the former gene being the most frequently found in clinical strains. However, their distribution frequency in environmental isolates is below 1%. Thus, very sensitive methods are required for detection and quantitation of tdh (+) strains in seafood. We previously reported a method to detect and quantify tdh (+) V. parahaemolyticus in seafood. This method consists of three components: the most-probable-number (MPN), the immunomagnetic separation (IMS) targeting all established K antigens, and the loop-mediated isothermal amplification (LAMP) targeting the tdh gene. However, this method faces regional issues in tropical zones of the world. Technicians have difficulties in securing dependable reagents in high-temperature climates where we found MPN underestimation in samples having tdh (+) strains as well as other microorganisms present at high concentrations. In the present study, we solved the underestimation problem associated with the salt polymyxin broth enrichment for the MPN component and with the immunomagnetic bead-target association for the IMS component. We also improved the supply and maintenance of the dependable reagents by introducing a dried reagent system to the LAMP component. The modified method is specific, sensitive, quick and easy and applicable regardless of the concentrations of tdh (+) V. parahaemolyticus. Therefore, we conclude this modified method is useful in world tropical, sub-tropical, and temperate zones.

  3. 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.

  4. DNA extraction from paraffin-embedded tissues using a salting-out procedure: a reliable method for PCR amplification of archival material.

    PubMed

    Howe, J R; Klimstra, D S; Cordon-Cardo, C

    1997-07-01

    Many techniques have been described for the extraction of DNA from paraffin-embedded tissues. Numerous efforts have been directed at simplification of these methods for rapid analysis using PCR. One disadvantage to some of the simpler procedures is inefficient PCR amplification, and for more involved ones using phenol/chloroform extraction, reduction in the yield of DNA. In the present study we report the use of a novel salting-out procedure that was utilized to extract DNA from 259 separate microdissection specimens of formalin-fixed, paraffin-embedded tissue sections. These sections were derived from 97 patients with tumors of the ampulla of Vater resected between 1965 and 1995 at our institution. The mean DNA yield was 22.75 micrograms (median 13.2 +/- 30.25) and the mean 260/280 absorbance ratio was 1.68 (median 1.70 +/- 0.25). All specimens (259/259) were successfully used to amplify K-ras exon 1 by a nested PCR technique. These results indicate that this DNA extraction method produces good yields of quality DNA, even from specimens several decades old.

  5. A PCR-based sex determination method for possible application in caprine gender selection by simultaneous amplification of the Sry and Aml-X genes.

    PubMed

    Phua, Alice Choon Yen; Abdullah, Ramli Bin; Mohamed, Zulqarnain

    2003-08-01

    Sex determination of livestock is performed to achieve the objectives of livestock breeding programmes. Techniques for sex determination have evolved from karyotyping to detecting Y-specific antigens and recently to the polymerase chain reaction (PCR), which appears to be the most sensitive, accurate, rapid and reliable method to date. In this study, a PCR-based sex determination method for potential application in goat breeding programmes was developed. Primers were designed to amplify a portion of the X amelogenin gene (Aml-X) on the X chromosome to give a 300 bp product and Sry gene on the Y chromosome to give a 116 bp product. PCR optimization was performed using DNA template extracted from a whole blood sample of Jermasia goats (German Fawn x Katjang) of both sexes. It was possible to identify the sex chromosomes by amplifying both male- and female-specific genes simultaneously in a duplex reaction with males yielding two bands and females yielding one band. The Aml-X primer set, which served as an internal control primer, did not interfere with amplification of the Y-specific sequence even when a low amount of DNA (1 ng) was used. The duplex reaction subjected to a blind test showed 100% (14/14) concordance, proving its accuracy and reliability. The primer sets used were found to be highly specific and were suitable for gender selection of goats.

  6. Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR.

    PubMed

    Suwancharoen, Duangjai; Sittiwicheanwong, Busara; Wiratsudakul, Anuwat

    2016-09-01

    Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle.

  7. Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR

    PubMed Central

    SUWANCHAROEN, Duangjai; SITTIWICHEANWONG, Busara; WIRATSUDAKUL, Anuwat

    2016-01-01

    Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop–mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen’s kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5–99.8) and 97.0% (95% CI 94.9–98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen’s kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle. PMID:27150208

  8. An improved method for detection of Edwardsiella tarda by loop-mediated isothermal amplification by targeting the EsrB gene

    NASA Astrophysics Data System (ADS)

    Xie, Guosi; Zhang, Qingli; Han, Nana; Shi, Chengyin; Wang, Xiuhua; Liu, Qinghui; Huang, Jie

    2012-07-01

    Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene ( EsrB — LAMP). In this method, the Mg2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene ( hemolysin -LAMP) for detection of E. tarda. The EsrB -LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples.

  9. A loop-mediated isothermal amplification (LAMP) assay for Strongyloides stercoralis in stool that uses a visual detection method with SYTO-82 fluorescent dye.

    PubMed

    Watts, Matthew R; James, Gregory; Sultana, Yasmin; Ginn, Andrew N; Outhred, Alexander C; Kong, Fanrong; Verweij, Jaco J; Iredell, Jonathan R; Chen, Sharon C-A; Lee, Rogan

    2014-02-01

    An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to < 10 copies of target sequence in a plasmid and up to a 10(-2) dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation.

  10. Development of loop-mediated isothermal amplification and SYBR green real-time PCR methods for the detection of Citrus yellow mosaic badnavirus in citrus species.

    PubMed

    Anthony Johnson, A M; Dasgupta, I; Sai Gopal, D V R

    2014-07-01

    Citrus yellow mosaic badnavirus (CMBV) is an important pathogen in southern India spread by infected citrus propagules. One of the measures to arrest the spread of CMBV is to develop methods to screen and certify citrus propagules as CMBV-free. The methods loop-mediated isothermal amplification (LAMP) and SYBR green real-time PCR (SGRTPCR) have been developed for the efficient detection of CMBV in citrus propagules. This paper compares the sensitivities of LAMP and SGRTPCR with polymerase chain reaction (PCR) for the detection of CMBV. Whereas PCR and LAMP were able to detect CMBV from a minimum of 10 ng of total DNA of infected leaf samples, SGRTPCR could detect the same from 1 ng of total DNA. Using SGRTPCR, the viral titres were estimated to be the highest in rough lemon and lowest in Nagpur Mandarin of the five naturally infected citrus species tested. The results will help in designing suitable strategies for the sensitive detection of CMBV from citrus propagules.

  11. Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR): a simple method for suppressing PCR amplification of specific DNA sequences using ORNs.

    PubMed

    Tanigawa, Naoki; Fujita, Toshitsugu; Fujii, Hodaka

    2014-01-01

    Polymerase chain reaction (PCR) amplification of multiple templates using common primers is used in a wide variety of molecular biological techniques. However, abundant templates sometimes obscure the amplification of minor species containing the same primer sequences. To overcome this challenge, we used oligoribonucleotides (ORNs) to inhibit amplification of undesired template sequences without affecting amplification of control sequences lacking complementarity to the ORNs. ORNs were effective at very low concentrations, with IC50 values for ORN-mediated suppression on the order of 10 nM. DNA polymerases that retain 3'-5' exonuclease activity, such as KOD and Pfu polymerases, but not those that retain 5'-3' exonuclease activity, such as Taq polymerase, could be used for ORN-mediated suppression. ORN interference-PCR (ORNi-PCR) technology should be a useful tool for both molecular biology research and clinical diagnosis.

  12. A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections

    PubMed Central

    Childs-Disney, Jessica L.; Disney, Matthew D.

    2008-01-01

    Herein, a method is described to increase the information density of sequencing experiments used to deconvolute nucleic acid selections. The method is facile and should be applicable to any selection experiment. A critical feature of this method is the use of biotinylated primers to amplify and encode a BamHI restriction site on both ends of a PCR product. After amplification, the PCR reaction is captured onto streptavidin resin, washed, and digested directly on the resin. Resin-based digestion affords clean product that is devoid of partially digested products and unincorporated PCR primers. The product's complementary ends are annealed and ligated together with T4 DNA ligase. Analysis of ligation products shows formation of concatemers of different length and little detectable monomer. Sequencing results produced data that routinely contained three to four copies of the library. This method allows for more efficient formulation of structure-activity relationships since multiple active sequences are identified from a single clone. PMID:18065718

  13. Validation of two methods for fatty acids analysis in eggs.

    PubMed

    Mazalli, Mônica R; Bragagnolo, Neura

    2007-05-01

    A comparative study between two methods (lipid extraction followed by saponification and methylation, and direct methylation) to determine the fatty acids in egg yolk was evaluated. Direct methylation of the samples resulted in lower fatty acid content and greater variation in the results than the lipid extraction followed by saponification and methylation. The low repeatability observed for the direct HCl methylation method was probably due to a less efficient extraction and conversion of the fatty acids into their methyl esters as compared to the same procedure starting with the lipid extract. As the lipid extraction followed by esterification method was shown to be more precise it was validated using powdered egg certified as reference material (RM 8415, NIST) and applied to samples of egg, egg enriched with polyunsaturated omega-3 fatty acids (n-3 PUFA), and commercial spray-dried whole egg powder.

  14. Fatty acids determination in Bronte pistachios by gas chromatographic method.

    PubMed

    Pantano, Licia; Lo Cascio, Giovanni; Alongi, Angelina; Cammilleri, Gaetano; Vella, Antonio; Macaluso, Andrea; Cicero, Nicola; Migliazzo, Aldo; Ferrantelli, Vincenzo

    2016-10-01

    A gas chromatographic with flame ionization detector (GC-MS FID) method for the identification and quantification of fatty acids based on the extraction of lipids and derivatisation of free acids to form methyl esters was developed and validated. The proposed method was evaluated to a number of standard FAs, and Bronte pistachios samples were used for that purpose and to demonstrate the applicability of the proposed method. In this regard, repeatability, mean and standard deviation of the analytical procedure were calculated. The results obtained have demonstrated oleic acid as the main component of Bronte pistachios (72.2%) followed by linoleic acid (13.4%) and showed some differences in composition with respect to Tunisian, Turkish and Iranian pistachios.

  15. Comparison of methods for acid quantification: impact of resist components on acid-generating efficiency

    NASA Astrophysics Data System (ADS)

    Cameron, James F.; Fradkin, Leslie; Moore, Kathryn; Pohlers, Gerd

    2000-06-01

    Chemically amplified deep UV (CA-DUV) positive resists are the enabling materials for manufacture of devices at and below 0.18 micrometer design rules in the semiconductor industry. CA-DUV resists are typically based on a combination of an acid labile polymer and a photoacid generator (PAG). Upon UV exposure, a catalytic amount of a strong Bronsted acid is released and is subsequently used in a post-exposure bake step to deprotect the acid labile polymer. Deprotection transforms the acid labile polymer into a base soluble polymer and ultimately enables positive tone image development in dilute aqueous base. As CA-DUV resist systems continue to mature and are used in increasingly demanding situations, it is critical to develop a fundamental understanding of how robust these materials are. One of the most important factors to quantify is how much acid is photogenerated in these systems at key exposure doses. For the purpose of quantifying photoacid generation several methods have been devised. These include spectrophotometric methods, ion conductivity methods and most recently an acid-base type titration similar to the standard addition method. This paper compares many of these techniques. First, comparisons between the most commonly used acid sensitive dye, tetrabromophenol blue sodium salt (TBPB) and a less common acid sensitive dye, Rhodamine B base (RB) are made in several resist systems. Second, the novel acid-base type titration based on the standard addition method is compared to the spectrophotometric titration method. During these studies, the make up of the resist system is probed as follows: the photoacid generator and resist additives are varied to understand the impact of each of these resist components on the acid generation process.

  16. A new method for selectively enhancing hemisphere processing: voice frequency amplification influences the strength of attribute framing.

    PubMed

    McCormick, Michael; Seta, John J

    2012-01-01

    An attribute framing effect occurs when positive or negative associations produced by positive or negative frames are mapped onto evaluations resulting in a more favourable evaluation for the positively framed attribute. We used a new voice frequency manipulation to differentially enhance right versus left hemisphere processing. In doing so we found a strong attribute framing effect when a speaker with a low-frequency voice enhanced the contextual processing style of the right hemisphere. However, a framing effect was not obtained when a speaker with a high-frequency voice enhanced the inferential/analytical processing style of the left hemisphere. At the theoretical level our results provide evidence that the contextual processing style of the right hemisphere is especially susceptible to associative implications, such as those found in attribute framing manipulations. At the applied level we provide a simple method for altering the effectiveness of persuasion messages.

  17. Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer.

    PubMed

    Iliadi, Alexandra; Petropoulou, Margarita; Ioannou, Penelope C; Christopoulos, Theodore K; Anagnostopoulos, Nikolaos I; Kanavakis, Emmanuel; Traeger-Synodinos, Jan

    2011-09-01

    In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.

  18. Two-stage cyclic enzymatic amplification method for ultrasensitive electrochemical assay of microRNA-21 in the blood serum of gastric cancer patients.

    PubMed

    Li, Bingchen; Liu, Fei; Peng, Yuanyuan; Zhou, Yunlei; Fan, Wenxuan; Yin, Huanshun; Ai, Shiyun; Zhang, Xiansheng

    2016-05-15

    MicroRNA (miRNA) is riveting nowadays due to its close relevance to human malignancies. Exploiting a fast and convenient biosensor for sensitive and specific detecting miRNA is necessary and it has significant meaning for oncology. In this work, to understand the relationship between miRNA-21 and gastric cancer, we employ T4 RNA ligase 2 to initiate specific ligation reaction depending on the sequence of target RNA, and T7 exonuclease to catalyze the first stage cyclic enzymatic amplification method (CEAM) specifically, which also resting on the target RNA sequence, and the second stage CEAM to further amplify the response resulting from the initial target RNA. Our two-stage CEAM strategy can detect miRNA-21 as low as 0.36fM with remarkable specificity, and most importantly, it can be used to inspect the expression level of miRNA-21 in blood serum of gastric cancer patients. This will offer new perspective for figuring out the pathways of miRNA-21 involving in cancer.

  19. Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.

    PubMed

    Yeh, Hung-Yueh; Shoemaker, Craig A; Klesius, Phillip H

    2005-10-01

    Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in 40--50 million dollars annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip 18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip 18 gene were also optimized at 65 degrees C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the E. ictaluri eip 18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.

  20. 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.

  1. Phenol-Sulfuric Acid Method for Total Carbohydrates

    NASA Astrophysics Data System (ADS)

    Nielsen, S. Suzanne

    The phenol-sulfuric acid method is a simple and rapid colorimetric method to determine total carbohydrates in a sample. The method detects virtually all classes of carbohydrates, including mono-, di-, oligo-, and polysaccharides. Although the method detects almost all carbohydrates, the absorptivity of the different carbohydrates varies. Thus, unless a sample is known to contain only one carbohydrate, the results must be expressed arbitrarily in terms of one carbohydrate.

  2. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    SciTech Connect

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2016-10-25

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacylethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings. The subject matter disclosed herein relates to enhancers of amidohydrolase activity.

  3. Method to produce succinic acid from raw hydrolysates

    DOEpatents

    Donnelly, Mark I.; Sanville-Millard, Cynthia Y.; Nghiem, Nhuan Phu

    2004-06-01

    A method for producing succinic acid from industrial-grade hydrolysates is provided, comprising supplying an organism that contains mutations for the genes ptsG, pflB, and ldhA, allowing said organism to accumulate biomass, and allowing said organism to metabolize the hydrolysate. Also provided is a bacteria mutant characterized in that it produces succinic acid from substrate contained in industrial-grade hydrolysate in a ratio of between 0.6:1 and 1.3:1 succinic acid to substrate.

  4. Determination of organic acids by CE and CEC methods.

    PubMed

    Klampfl, Christian W

    2007-10-01

    A comprehensive overview of the analysis of low-molecular-mass organic acids employing electromigration methods in the capillary format is given. This review includes papers published since 2003 and can be seen as an update of the review paper published by Galli et al. in 2003. Tables included in this review contain application papers describing the determination of organic acids from a variety of fields like the analysis of food and beverages, environmental samples, samples from clinical origin, and from natural products.

  5. 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.

  6. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

    PubMed

    Yang, Qi; Franco, Christopher M M; Zhang, Wei

    2015-10-01

    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

  7. Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction

    PubMed Central

    Mok, Ellie; Wee, Eugene; Wang, Yuling; Trau, Matt

    2016-01-01

    The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community. PMID:27910874

  8. 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

  9. Evaluation of acidity estimation methods for mine drainage, Pennsylvania, USA.

    PubMed

    Park, Daeryong; Park, Byungtae; Mendinsky, Justin J; Paksuchon, Benjaphon; Suhataikul, Ratda; Dempsey, Brian A; Cho, Yunchul

    2015-01-01

    Eighteen sites impacted by abandoned mine drainage (AMD) in Pennsylvania were sampled and measured for pH, acidity, alkalinity, metal ions, and sulfate. This study compared the accuracy of four acidity calculation methods with measured hot peroxide acidity and identified the most accurate calculation method for each site as a function of pH and sulfate concentration. Method E1 was the sum of proton and acidity based on total metal concentrations; method E2 added alkalinity; method E3 also accounted for aluminum speciation and temperature effects; and method E4 accounted for sulfate speciation. To evaluate errors between measured and predicted acidity, the Nash-Sutcliffe efficiency (NSE), the coefficient of determination (R (2)), and the root mean square error to standard deviation ratio (RSR) methods were applied. The error evaluation results show that E1, E2, E3, and E4 sites were most accurate at 0, 9, 4, and 5 of the sites, respectively. Sites where E2 was most accurate had pH greater than 4.0 and less than 400 mg/L of sulfate. Sites where E3 was most accurate had pH greater than 4.0 and sulfate greater than 400 mg/L with two exceptions. Sites where E4 was most accurate had pH less than 4.0 and more than 400 mg/L sulfate with one exception. The results indicate that acidity in AMD-affected streams can be accurately predicted by using pH, alkalinity, sulfate, Fe(II), Mn(II), and Al(III) concentrations in one or more of the identified equations, and that the appropriate equation for prediction can be selected based on pH and sulfate concentration.

  10. The evaluation method for antiplatelet effect of acetylsalicylic acid.

    PubMed

    Yokoyama, Haruko; Mastumura, Takashi; Soeda, Shinji; Suzuki, Yuji; Watanabe, Masayuki; Kashiwakura, Emiko; Saso, Takayuki; Ikeda, Noriyuki; Tokuoka, Kentaro; Kitagawa, Yasuhisa; Yamada, Yasuhiko

    2014-12-01

    Reduced platelet aggregation by acetylsalicylic acid administration has been associated with adverse outcomes in patients with thrombotic diseases, thus it is important to determine aspirin resistance in those cases. The antiplatelet effect of acetylsalicylic acid is rarely measured, but it has many problems. The aim of this study was to find the evaluation method for antiplatelet effect after administration of acetylsalicylic acid. We developed a particle counting method based upon laser light scattering, and utilized the platelet aggregation agonists, collagen, at 0.25, 0.5 and 1.0 μg/mL, and adenosine diphosphate (ADP), at 0.5, 1.0 and 2.0 μM, to determine their effective concentrations. Seventeen healthy volunteers were administered acetylsalicylic acid at 162 mg/day, with platelet aggregation determined before and 20 min after administration. In all subjects, the rate of platelet aggregation induced by 1.0 μg/mL of collagen before taking acetylsalicylic acid was the highest value obtained, while 20 min after acetylsalicylic acid administration, aggregation induced by collagen at 1.0 μg/mL was significantly decreased as compared to before administration. As for the other concentrations of collagen and all those of ADP tested, platelet aggregation was either not significantly induced before taking acetylsalicylic acid or the rate of aggregation was not significantly decreased after taking acetylsalicylic acid. Our results indicate that collagen at 1.0 μg/mL is appropriate as a platelet aggregation agonist for evaluating the antiplatelet effect of acetylsalicylic acid. Thus, it is useful that the measurement is performed only once.

  11. Methods And Devices For Characterizing Duplex Nucleic Acid Molecules

    DOEpatents

    Akeson, Mark; Vercoutere, Wenonah; Haussler, David; Winters-Hilt, Stephen

    2005-08-30

    Methods and devices are provided for characterizing a duplex nucleic acid, e.g., a duplex DNA molecule. In the subject methods, a fluid conducting medium that includes a duplex nucleic acid molecule is contacted with a nanopore under the influence of an applied electric field and the resulting changes in current through the nanopore caused by the duplex nucleic acid molecule are monitored. The observed changes in current through the nanopore are then employed as a set of data values to characterize the duplex nucleic acid, where the set of data values may be employed in raw form or manipulated, e.g., into a current blockade profile. Also provided are nanopore devices for practicing the subject methods, where the subject nanopore devices are characterized by the presence of an algorithm which directs a processing means to employ monitored changes in current through a nanopore to characterize a duplex nucleic acid molecule responsible for the current changes. The subject methods and devices find use in a variety of applications, including, among other applications, the identification of an analyte duplex DNA molecule in a sample, the specific base sequence at a single nulceotide polymorphism (SNP), and the sequencing of duplex DNA molecules.

  12. A mixture detection method based on separate amplification using primer specific alleles of INDELs-a study based on two person's DNA mixture.

    PubMed

    Liu, Jinding; Wang, Jiaqi; Zhang, Xiaojia; Li, Zeqin; Yun, Keming; Liu, Zhizhen; Zhang, Gengqian

    2017-02-01

    Samples containing unbalanced DNA mixtures from individuals often occur in forensic DNA examination and clinical detection. Because of the PCR amplification bias, the minor contributor DNA is often masked by the major contributor DNA when using traditional STR or SNP typing techniques. Here we propose a method based in allele-specific Insertion/Deletion (INDEL) genotyping to detect DNA mixtures in forensic samples. Fourteen INDELs were surveyed in the Chinese Han population of Shanxi Province. The INDELs were amplified using two separate primer-specific reactions by real-time PCR. The difference Ct value of the 2 reactions (D-value) were used for determination of the single source DNA. INDELs types and further confirmed by electrophoresis separation. The minor allele frequency (MAF) was above 0.2 in 10 INDELs. The detection limit was 0.3125 ng-1.25 ng template DNA for real-time PCR in all 14 INDEL markers. For single source 10 ng DNA, the average D-value was 0.31 ± 0.14 for LS type, 6.96 ± 1.05 for LL type and 7.20 ± 1.09 for SS type. For the series of simulated DNA mixture, the Ct value varied between the ranges of single source DNA, depending on their INDEL typing and mixture ratios. This method can detect the specific allele of the minor DNA contributor as little as 1:50 in rs397782455 and rs397696936; 1:100 in rs397832665, rs397822382 and rs397897230; the detection limit of the minor DNA contributor was as little as 1:500-1:1000 in the rest INDEL markers, a much higher sensitivity compared with traditional STR typing. The D-value variation depended on the alternation of dilution ratio and INDEL types. When the dilution was 1:1000, the maximum and minimum D-values were 8.84 ± 0.11 in rs397897230 and 4.27 ± 0.19 in rs397897239 for LL and SS type mixture, the maximum and minimum D-values were 9.32 ± 0.54 in rs397897230 and 4.38 ± 0.26 in rs 397897239 for LL(SS) and LS type mixture, separately. Any D-value between 0.86 and 5.11 in the 14

  13. Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

    DOEpatents

    Weier, Heinz-Ulrich G.; Gray, Joe W.

    1995-01-01

    A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers ard probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity.

  14. Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

    DOEpatents

    Weier, H.U.G.; Gray, J.W.

    1995-06-27

    A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers and probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity. 18 figs.

  15. 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.

  16. A method to attenuate U(VI) mobility in acidic waste plumes using humic acids

    SciTech Connect

    Wan, J.; Dong, W.; Tokunaga, T.K.

    2011-02-01

    Acidic uranium (U) contaminated plumes have resulted from acid-extraction of plutonium during the Cold War and from U mining and milling operations. A sustainable method for in-situ immobilization of U under acidic conditions is not yet available. Here, we propose to use humic acids (HAs) for in-situ U immobilization in acidic waste plumes. Our laboratory batch experiments show that HA can adsorb onto aquifer sediments rapidly, strongly and practically irreversibly. Adding HA greatly enhanced U adsorption capacity to sediments at pH below 5.0. Our column experiments using historically contaminated sediments from the Savannah River Site under slow flow rates (120 and 12 m/y) show that desorption of U and HA were non-detectable over 100 pore-volumes of leaching with simulated acidic groundwaters. Upon HA-treatment, 99% of the contaminant [U] was immobilized at pH < 4.5, compared to 5% and 58% immobilized in the control columns at pH 3.5 and 4.5, respectively. These results demonstrated that HA-treatment is a promising in-situ remediation method for acidic U waste plumes. As a remediation reagent, HAs are resistant to biodegradation, cost effective, nontoxic, and easily introducible to the subsurface.

  17. Comparative assessment of the methods for exchangeable acidity measuring

    NASA Astrophysics Data System (ADS)

    Vanchikova, E. V.; Shamrikova, E. V.; Bespyatykh, N. V.; Zaboeva, G. A.; Bobrova, Yu. I.; Kyz"yurova, E. V.; Grishchenko, N. V.

    2016-05-01

    A comparative assessment of the results of measuring the exchangeable acidity and its components by different methods was performed for the main mineral genetic horizons of texturally-differentiated gleyed and nongleyed soddy-podzolic and gley-podzolic soils of the Komi Republic. It was shown that the contents of all the components of exchangeable soil acidity determined by the Russian method (with potassium chloride solution as extractant, c(KCl) = 1 mol/dm3) were significantly higher than those obtained by the international method (with barium chloride solution as extractant, c(BaCl2) = 0.1 mol/dm3). The error of the estimate of the concentration of H+ ions extracted with barium chloride solution equaled 100%, and this allowed only qualitative description of this component of the soil acidity. In the case of the extraction with potassium chloride, the error of measurements was 50%. It was also shown that the use of potentiometric titration suggested by the Russian method overestimates the results of soil acidity measurement caused by the exchangeable metal ions (Al(III), Fe(III), and Mn(II)) in comparison with the atomic emission method.

  18. A GC-ECD method for estimation of free and bound amino acids, gamma-aminobutyric acid, salicylic acid, and acetyl salicylic acid from Solanum lycopersicum (L.).

    PubMed

    Meher, Hari Charan; Gajbhiye, Vijay T; Singh, Ghanendra

    2011-01-01

    A gas chromatograph with electron capture detection method for estimation of selected metabolites--amino acids (free and bound), gamma-aminobutyric acid (GABA), salicylic acid (SA), and acetyl salicylic acid (ASA) from tomato--is reported. The method is based on nitrophenylation of the metabolites by 1-fluoro-2, 4-dinitrobenzene under aqueous alkaline conditions to form dinitophenyl derivatives. The derivatives were stable under the operating conditions of GC. Analysis of bound amino acids comprised perchloric acid precipitation of protein, alkylation (carboxymethylation) with iodoacetic acid, vapor-phase hydrolysis, and derivatization with 1-fluoro-2,4-dinitrobenzene in that order. The metabolites were resolved in 35 min, using a temperature-programmed run. The method is rapid, sensitive, and precise. It easily measured the typical amino acids (aspartate, asparagine, glutamate, glutamine, alanine, leucine, lysine, and phenylalanine) used for identification and quantification of a protein, resolved amino acids of the same mass (leucine and isoleucine), satisfactorily measured sulfur amino acid (methionine, cystine, and cysteine), and quantified GABA, SA, and ASA, as well. The developed method was validated for specificity, linearity, and precision. It has been applied and recommended for estimation of 25 metabolites from Solanum lycopersicum (L.).

  19. A nationwide survey of pathogenic leptospires in urine of cattle and buffaloes by Loop-mediated isothermal amplification (LAMP) method in Thailand, 2011–2013

    PubMed Central

    SUWANCHAROEN, Duangjai; LIMLERTVATEE, Supaluck; CHETIYAWAN, Philaiphon; TONGPAN, Phichet; SANGKAEW, Nongluck; SAWADDEE, Yaowarat; INTHAKAN, Kanya; WIRATSUDAKUL, Anuwat

    2016-01-01

    Leptospirosis is a worldwide distributed zoonosis which has long been endemic in Thailand. Cattle and buffaloes are important livestock species that live in close contact with humans, especially in rural areas. These animals may, therefore, act as long-term carriers of leptospirosis for humans and other livestock species. The present study employed loop-mediated isothermal amplification (LAMP) method to detect pathogenic leptospiral 16S rDNA in the urine of cattle and buffaloes for assessing associations between uroprevalence and species, sex, age and spatial distribution. A total of 3,657 urine samples were collected for laboratory diagnosis, and 312 of which turned positive to the test (true prevalence 5.90%; 95% CI 4.98–6.91). The highest true uroprevalence was found in lower northern region at 19.80% (95% CI 15.83–24.32) followed by upper and lower northeastern regions at 15.22% and 6.25%, respectively. However, the highest true uroprevalence in beef cattle, the majority of cattle in Thailand, was recorded in northeastern region which is the endemic area of human leptospirosis. The uroprevalence was not statistically different among species and types of examined animals. Male animals were over twice more likely to be infected compared to females. Excluding animals younger than one year of age due to small sample size, the uroprevalence upraised with increasing age. A collaborative investigation between veterinary and public health sectors is required to holistically explore the link between leptospirosis in humans and livestock, especially in high prevalent areas. PMID:27302016

  20. Roka Listeria detection method using transcription mediated amplification to detect Listeria species in select foods and surfaces. Performance Tested Method(SM) 011201.

    PubMed

    Hua, Yang; Kaplan, Shannon; Reshatoff, Michael; Hu, Ernie; Zukowski, Alexis; Schweis, Franz; Gin, Cristal; Maroni, Brett; Becker, Michael; Wisniewski, Michele

    2012-01-01

    The Roka Listeria Detection Assay was compared to the reference culture methods for nine select foods and three select surfaces. The Roka method used Half-Fraser Broth for enrichment at 35 +/- 2 degrees C for 24-28 h. Comparison of Roka's method to reference methods requires an unpaired approach. Each method had a total of 545 samples inoculated with a Listeria strain. Each food and surface was inoculated with a different strain of Listeria at two different levels per method. For the dairy products (Brie cheese, whole milk, and ice cream), our method was compared to AOAC Official Method(SM) 993.12. For the ready-to-eat meats (deli chicken, cured ham, chicken salad, and hot dogs) and environmental surfaces (sealed concrete, stainless steel, and plastic), these samples were compared to the U.S. Department of Agriculture/Food Safety and Inspection Service-Microbiology Laboratory Guidebook (USDA/FSIS-MLG) method MLG 8.07. Cold-smoked salmon and romaine lettuce were compared to the U.S. Food and Drug Administration/Bacteriological Analytical Manual, Chapter 10 (FDA/BAM) method. Roka's method had 358 positives out of 545 total inoculated samples compared to 332 positive for the reference methods. Overall the probability of detection analysis of the results showed better or equivalent performance compared to the reference methods.

  1. Method for preparing 6-.beta.-halopenicillanic acids

    DOEpatents

    Hansen, Erik I.; Kran-Nielsen, Mogens P.; Von Daehne, Welf

    1989-01-01

    The present invention relates to a new and improved method for the preparation of a compound of the formula I ##STR1## in which R stands for halogen, giving rise to high yields of substantially pure 6.beta.-halopenicillanic acids, obtained in one step.

  2. An analytical method for the measurement of acid metabolites of tryptophan-NAD pathway and related acids in urine.

    PubMed

    Liao, Xiangjun; Zhu, Jiping; Rubab, Mamoona; Feng, Yong-Lai; Poon, Raymond

    2010-04-15

    An analytical method has been developed for the measurements of five urinary acids namely, quinolinic acid, picolinic acid, nicotinic acid, 2-pyridylacetic acid and 3-pyridylacetic acid. The high performance liquid chromatograph-electrospray ionization mass spectrometry was operated in positive polarity under selected ion monitoring mode, with a column flow rate of 0.2 ml/min and an injection volume of 20 microl. The method used isotope-labelled picolinic acid (PA-d(4)) and nicotinic acid (NA-d(4)) as internal standards for the quantification. The sample preparation involved parallel use of two different types of mixed-mode solid phase extraction cartridges (Strata-X-AW for the extraction of quinolinic acid, and Strata-X-C for the remaining acids). Quantitative analysis of five target acids in several human and rat urine samples showed that the levels of acids were relatively uniform among rats while larger variations were observed for human samples.

  3. A simplified method for estimation of jarosite and acid-forming sulfates in acid mine wastes.

    PubMed

    Li, Jun; Smart, Roger St C; Schumann, Russell C; Gerson, Andrea R; Levay, George

    2007-02-01

    In acid base accounting (ABA) estimates of acid mine wastes, the acid potential (AP) estimate can be improved by using the net carbonate value (NCV) reactive sulfide S method rather than total S assay methods but this does not give recovery of potentially acid producing ferrous and ferric sulfates present in many wastes. For more accurate estimation of AP, an effective, site-specific method to quantify acid sulfate salts, such as jarosite and melanterite, in waste rocks has been developed and tested on synthetic and real wastes. The SPOCAS (acid sulfate soils) methods have been modified to an effective, rapid method to speciate sulfate forms in different synthetic waste samples. A three-step sequential extraction procedure has been established. These steps are: (1) argon-purged water extraction (3 min) to extract soluble Fe(II) salts (particularly melanterite), epsomite and gypsum (<10 wt.%), (2) roasting at 550 degrees C (1 h) to remove sulfur from pyrite and other reactive sulfides, (3) HCl extraction (4 M, 30 min) for determination of jarosites. Products (solid and aqueous) have been characterized at each step including the jarosite decomposition process in Step 2 where temperature control is critical to avoid S loss. The sequential extraction procedure was used to quantitatively determine melanterite, epsomite, gypsum, pyrite and jarosite concentrations in a synthetic waste sample containing these mineral phases at 5 wt.% in quartz, and also tested using a tailings waste sample to quantitatively determine epsomite, gypsum and jarosite contents. The method is applicable to most waste samples including those with non-pyrite sulfides but for samples containing significant amounts of sulfur (>1 wt.% S) as copper sulfides, the second step of roasting needs to be excluded from the procedure with an increased time of 4 M HCl extraction to 16 h for jarosite determination.

  4. 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.

  5. 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...

  6. Determination of L- Ascorbic Acid in Plasma by Voltammetric Method

    PubMed Central

    Behfar, Abdol Azim; Sadeghi, Nafiseh; Jannat, Behrooz; Oveisi, Mohammad Reza

    2010-01-01

    Voltammetric techniques have been considered as important methods among the analytical techniques used for the identification and determination of trace concentrations of many biological molecules such as L-ascorbic acid (AA). L-ascorbic acid is an electro-active molecule, though it is difficult to determine its value directly with a majority of electrodes made of carbon and transition metals, because of electrode surface problems. The present study is based on I-E curves for AA analysis at various pH. Furthermore, the effects of the presence of other electro-active substances; such as copper, as well as the effect of the sweep rate of potential will be studied. The present study is based on analysis of the current-voltage curves for L-ascorbic acid at varying pH and sweep rate scan values. An analysis was also carried out to measure the influence of the concentration of some electro active species. The peak height of the first oxidation wave is used for L-ascorbic acid assay. L-ascorbic acid was determined in aqueous media by linear-scan voltammetry on a gold electrode; ranging between (1-175 μg/mL). In biologic samples, for elimination of uric acid or some sugars and effects, a significant interference of copper ions whose presence reduces the height of the L-ascorbic acid oxidation peak was used. The optimum pH and sweep rate were 3.2 and 7500mV/s, respectively. Under these conditions, the detection limit of the method was 0.3 μg/mL. Repeatability of the method based on relative standard deviation (RSD) 50, 10 and 1 μg/mL concentrations was 0.83, 2.1 and 10.3%, respectively. The calibration curve was linear over the range 1-175μg/mL (r2 = 0.9977, p < 0.001). The advantage of this method lies in the fact that the use of copper eliminates the interference of different substances such as uric acid. PMID:24363717

  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

    important framework for the integration of QD-FRET methods with digital imaging for a ratiometric transduction of nucleic acid hybridization on a paper-based platform.

  8. 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.

  9. Method comparison study for weak acid dissociation cyanide analysis.

    PubMed

    Evans, Joseph D; Thompson, Leslie; Clark, Patrick J; Beckman, Scott W

    2003-02-01

    Method comparison studies of two different methods for the analysis of weak acid dissociable (WAD) cyanide revealed analytical flaws and/or matrix interference problems with both procedures. EPA "draft" method 1677 using a Perstorp 3202 CN analyzer was compared to Standard Method 4500 CN I. It was discovered that the Perstorp analyzer produced more precise and more accurate results once appropriate and necessary procedural steps from the EPA draft method were modified. Comparison of these two methods, was based on "real world" samples collected from a mine-tailing solution. The mine-tailing solution contained high concentrations of cyanide and metals. Inconsistencies in method procedures were traced to sulfide interferences and high concentrations of WAD metals. Conclusions were based upon a large sample base collected from a mine site over a 90-day period.

  10. 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.

  11. Acid pre-treatment method for in situ ore leaching

    DOEpatents

    Mallon, R.G.; Braun, R.L.

    1975-10-28

    An acid leaching method is described for the recovery of a desired element from a subterranean rubblized body of primary ore containing the element and also having associated therewith a carbonate mineral wherein the rubblized ore body is flooded with an aqueous acidic solution in order to release carbon dioxide from the associated carbonate mineral. After a substantial portion of the available carbon dioxide is released and removed from the ore body, as by venting to the atmosphere, an oxidizing gas is introduced into the flooded, rubblized ore to oxidize the ore and form an acid leach solution effective in the presence of the dissolved oxidizing gas to dissolve the ore and cause the desired element to go into solution. The leach solution is then circulated to the surface where the metal values are recovered therefrom.

  12. 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.)

  13. Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA.

    PubMed

    Turchinovich, Andrey; Surowy, Harald; Serva, Andrius; Zapatka, Marc; Lichter, Peter; Burwinkel, Barbara

    2014-01-01

    Massive parallel sequencing (MPS) technologies have paved the way into new areas of research including individualized medicine. However, sequencing of trace amounts of DNA or RNA still remains a major challenge, especially for degraded nucleic acids like circulating DNA. This together with high cost and time requirements impedes many important applications of MPS in medicine and fundamental science. We have established a fast, cheap and highly efficient protocol called 'Capture and Amplification by Tailing and Switching' (CATS) to directly generate ready-to-sequence libraries for MPS from nanogram and picogram quantities of both DNA and RNA. Furthermore, those DNA libraries are strand-specific, can be prepared within 2-3 h and do not require preliminary sample amplification steps. To exemplify the capacity of the technique, we have generated and sequenced DNA libraries from hundred-picogram amounts of circulating nucleic acids isolated from human blood plasma, one nanogram of mRNA-enriched total RNA from cultured cells and few nanograms of bisulfite-converted DNA. The approach for DNA library preparation from minimal and fragmented input described here will find broad application in diverse research areas such as translational medicine including therapy monitoring, prediction, prognosis and early detection of various human disorders and will permit high-throughput DNA sequencing from previously inaccessible material such as minute forensic and archeological samples.

  14. Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques.

    PubMed

    Neumaier, M; Braun, A; Wagener, C

    1998-01-01

    The increasing interest in molecular biology diagnostics is a result of the tremendous gain of scientific knowledge in genetics, made possible especially since the introduction of amplification techniques. High expectations have been placed on genetic testing, and the number of laboratories now using the relevant technology is rapidly increasing--resulting in an obvious need for standardization and definition of laboratory organization. This communication is an effort towards that end. We address aspects that should be considered when structuring a new molecular diagnostic laboratory, and we discuss individual preanalytical and analytical procedures, from sampling to evaluation of assay results. In addition, different means of controlling contamination are discussed. Because the methodology is in constant change, no general standards can be defined. Accordingly, this publication is intended to serve as a recommendation for good laboratory practice and internal quality control and as a guide to troubleshooting, primarily in amplification techniques.

  15. Improved RNA quality and TaqMan® Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials

    PubMed Central

    Li, Jinghuan; Smyth, Paul; Cahill, Susanne; Denning, Karen; Flavin, Richard; Aherne, Sinead; Pirotta, Marco; Guenther, Simone M; O'Leary, John J; Sheils, Orla

    2008-01-01

    Background Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62–164 bp). Results Results from the Bioanalyzer and TaqMan® data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70°C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan® detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan® PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification. Conclusion Modification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan® PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts. PMID:18254955

  16. Method for liquid chromatographic extraction of strontium from acid solutions

    DOEpatents

    Horwitz, E. Philip; Dietz, Mark L.

    1992-01-01

    A method and apparatus for extracting strontium and technetium values from biological, industrial and environmental sample solutions using a chromatographic column is described. An extractant medium for the column is prepared by generating a solution of a diluent containing a Crown ether and dispersing the solution on a resin substrate material. The sample solution is highly acidic and is introduced directed to the chromatographic column and strontium or technetium is eluted using deionized water.

  17. Method and apparatus for staining immobilized nucleic acids

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.; Jacobson, Stephen C.

    2000-01-01

    A method for staining immobilized nucleic acids includes the steps of affixing DNA probes to a solid substrate, moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes, and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

  18. 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

  19. 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.

  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

    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.

  1. Properties of nanocellulose isolated from corncob residue using sulfuric acid, formic acid, oxidative and mechanical methods.

    PubMed

    Liu, Chao; Li, Bin; Du, Haishun; Lv, Dong; Zhang, Yuedong; Yu, Guang; Mu, Xindong; Peng, Hui

    2016-10-20

    In this work, nanocellulose was extracted from bleached corncob residue (CCR), an underutilized lignocellulose waste from furfural industry, using four different methods (i.e. sulfuric acid hydrolysis, formic acid (FA) hydrolysis, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, and pulp refining, respectively). The self-assembled structure, morphology, dimension, crystallinity, chemical structure and thermal stability of prepared nanocellulose were investigated. FA hydrolysis produced longer cellulose nanocrystals (CNCs) than the one obtained by sulfuric acid hydrolysis, and resulted in high crystallinity and thermal stability due to its preferential degradation of amorphous cellulose and lignin. The cellulose nanofibrils (CNFs) with fine and individualized structure could be isolated by TEMPO-mediated oxidation. In comparison with other nanocellulose products, the intensive pulp refining led to the CNFs with the longest length and the thickest diameter. This comparative study can help to provide an insight into the utilization of CCR as a potential source for nanocellulose production.

  2. 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.

  3. Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

    PubMed

    Glais, Laurent; Jacquot, Emmanuel

    2015-01-01

    Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

  4. Heat induces gene amplification in cancer cells

    SciTech Connect

    Yan, Bin; Ouyang, Ruoyun; Huang, Chenghui; Liu, Franklin; Neill, Daniel; Li, Chuanyuan; Dewhirst, Mark

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  5. 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.

  6. Mycolic Acid Index Susceptibility Method for Mycobacterium tuberculosis

    PubMed Central

    Viader-Salvadó, José M.; Garza-González, Elvira; Valdez-Leal, Ramón; de los Angeles del Bosque-Moncayo, M.; Tijerina-Menchaca, Rolando; Guerrero-Olazarán, Martha

    2001-01-01

    A rapid drug susceptibility test to measure the susceptibility of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) using clinical isolates and a newly defined mycolic acid index (MAI) was evaluated. A total of 200 clinical isolates of M. tuberculosis were tested for susceptibility or resistance to INH and RIF by the MAI susceptibility and indirect-proportion methods. Overall, there was agreement between the two methods for 398 (99.5%) of the 400 total tests. Specifically, the sensitivity of the MAI susceptibility method for INH and RIF was 97.6 and 100%, respectively. The specificity and positive predictive value were 100% for both drugs, and the negative predictive value for INH and RIF was 98.3 and 100%, respectively. In conclusion, the MAI susceptibility method described here can be used for rapid drug susceptibility testing of M. tuberculosis clinical isolates within 5 days after clinical isolates are incubated in the presence or absence of an antituberculosis drug. PMID:11427584

  7. A microarray-based method to perform nucleic acid selections.

    PubMed

    Aminova, Olga; Disney, Matthew D

    2010-01-01

    This method describes a microarray-based platform to perform nucleic acid selections. Chemical ligands to which a nucleic acid binder is desired are immobilized onto an agarose microarray surface; the array is then incubated with an RNA library. Bound RNA library members are harvested directly from the array surface via gel excision at the position on the array where a ligand was immobilized. The RNA is then amplified via RT-PCR, cloned, and sequenced. This method has the following advantages over traditional resin-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX): (1) multiple selections can be completed in parallel on a single microarray surface; (2) kinetic biases in the selections are mitigated since all RNA binders are harvested from an array via gel excision; (3) the amount of chemical ligand needed to perform a selection is minimized; (4) selections do not require expensive resins or equipment; and (5) the matrix used for selections is inexpensive and easy to prepare. Although this protocol was demonstrated for RNA selections, it should be applicable for any nucleic acid selection.

  8. Hydroxyapatite-phosphonoformic acid hybrid compounds prepared by hydrothermal method

    NASA Astrophysics Data System (ADS)

    Turki, Thouraya; Othmani, Masseoud; Bantignies, Jean-Louis; Bouzouita, Khaled

    2014-01-01

    Hydroxyapatites were prepared in the presence of different amounts of phosphonoformic acid (PFA) via the hydrothermal method. The obtained powders were characterized through chemical analysis, XRD, IR, 31P MAS-NMR, TEM, and TG-TDA. The XRD showed that the PFA did not affect the apatite composition. Indeed, only a reduction of the crystallite size was noted. After grafting of PFA, the IR spectroscopy revealed the appearance of new bands belonging to HPO42- and carboxylate groups of the apatite and organic moiety, respectively. Moreover, the 31P MAS-NMR spectra exhibited a peak with a low intensity assigned to the terminal phosphonate group of the organic moiety in addition to that of the apatite. Based on these results, a reaction mechanism involving the surface hydroxyl groups (tbnd Casbnd OH) of the apatite and the carboxyl group of the acid was proposed.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction.

    PubMed

    Sagar, Kalpana; Singh, Salam Pradeep; Goutam, Kapil Kumar; Konwar, Bolin Kumar

    2014-02-01

    Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was >23 kb and yield 0.5-5 μg/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500 bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction.

  14. Method for the isolation of citric acid and malic acid in Japanese apricot liqueur for carbon stable isotope analysis.

    PubMed

    Akamatsu, Fumikazu; Hashiguchi, Tomokazu; Hisatsune, Yuri; Oe, Takaaki; Kawao, Takafumi; Fujii, Tsutomu

    2017-02-15

    A method for detecting the undeclared addition of acidic ingredients is required to control the authenticity of Japanese apricot liqueur. We developed an analytical procedure that minimizes carbon isotope discrimination for measurement of the δ(13)C values of citric and malic acid isolated from Japanese apricot liqueur. Our results demonstrated that freeze-drying is preferable to nitrogen spray-drying, because it does not significantly affect the δ(13)C values of citric acid and results in smaller isotope discrimination for malic acid. Both 0.1% formic acid and 0.2% phosphoric acid are acceptable HPLC mobile phases for the isolation of citric and malic acid, although the δ(13)C values of malic acid exhibited relatively large variation compared with citric acid following isolation using either mobile phase. The developed procedure allows precise δ(13)C measurements of citric and malic acid isolated from Japanese apricot liqueur.

  15. Rapid Detection of Subtype H10N8 Influenza Virus by One-Step Reverse Transcription–Loop-Mediated Isothermal Amplification Methods

    PubMed Central

    Bao, Hongmei; Feng, Xiaoxiao; Ma, Yong; Shi, Jianzhong; Zhao, Yuhui; Gu, Linlin

    2015-01-01

    We developed hemagglutinin- and neuraminidase-specific one-step reverse transcription–loop-mediated isothermal amplification assays for detecting the H10N8 virus. The detection limit of the assays was 10 copies of H10N8 virus, and the assays did not amplify nonspecific RNA. The assays can detect H10N8 virus from chicken samples with high sensitivity and specificity, and they can serve as an effective tool for detecting and monitoring H10N8 virus in live poultry markets. PMID:26378283

  16. The Discovery of Rolling Circle Amplification and Rolling Circle Transcription.

    PubMed

    Mohsen, Michael G; Kool, Eric T

    2016-11-15

    even without promoter sequences in the circle. We describe how one can encode cleavable sites into the product DNAs and RNAs from RCA/RCT, which can then be resolved into large quantities of almost pure oligonucleotides. Our Account then proceeds with a summary describing a broad variety of tools and methods built in many laboratories around the rolling circle concept. Among the important developments are the discovery of highly efficient DNA polymerases for RCA; the invention of exponential ("hyperbranched") RCA amplification made possible by use of a second primer; the development of the "padlock" process for detection of nucleic acids and proteins coupled with RCA; the use of circular oligonucleotides as vectors in cells to encode biologically active RNAs via RCT; and the use of small DNA circles to encode and extend human telomeres. Finally, we finish with some ideas about where the field may go in the future.

  17. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    PubMed Central

    Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

    2015-01-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20–100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device. PMID:26050646

  18. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    NASA Astrophysics Data System (ADS)

    Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

    2015-06-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.

  19. Rapid isothermal detection of Phytophthora species on plant samples using recombinase polymerase amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently several isothermal amplification techniques have been developed that are extremely tolerant towards inhibitors present in many plant extracts. Recombinase polymerase amplification (RPA) assays for the genus Phytophthora have been developed which provide a simple and rapid method to macerate...

  20. 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.

  1. 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

  2. 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.

  3. Y chromosome specific nucleic acid probe and method for identifying the Y chromosome in SITU

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich

    1999-01-01

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences.

  4. Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

    DOEpatents

    Gray, J.W.; Weier, H.U.

    1998-11-24

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences. 9 figs.

  5. Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich

    1998-01-01

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences.

  6. Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich

    2001-01-01

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences.

  7. Y chromosome specific nucleic acid probe and method for identifying the Y chromosome in SITU

    DOEpatents

    Gray, J.W.; Weier, H.U.

    1999-03-30

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences. 9 figs.

  8. 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

  9. 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.

  10. Simple method of isolating humic acids from organic soils

    NASA Astrophysics Data System (ADS)

    Ahmed, O.

    2009-04-01

    Humic substances particularly humic acids (HA) play a major role in soil conditioning e.g. erosion control, soil cation exchange capacity, complexation of heavy metal ions and pesticides, carbon and nitrogen cycles, plant growth and reduction of ammonia volatilization from urea. Humified substances such as coal, composts, and peat soils have substantial amounts of HA but the isolation of these acids is expensive, laborious, and time consuming. Factors that affect the quality and yield of HA isolated from these materials include extraction, fractionation, and purification periods. This work developed a simple, rapid, and cost effective method of isolating HA from peat soils. There was a quadratic relationship between extraction period and HA yield. Optimum extraction period was estimated at 4 h instead of the usual range of 12 to 48 h. There was no relationship between fractionation period and HA yield. As such 2 h instead of the usual range of 12 to 24 h fractionation period could be considered optimum. Low ash content (5%), remarkable reduction in K, coupled with the fact that organic C, E4/E6, carboxylic COOH, phenolic OH, and total acidity values of the HA were consistent with those reported by other authors suggest that the HA dealt with were free from mineral matter. This was possible because the distilled water used to purify the HA served as Bronsted-Lowry acid during the purification process of the HA. Optimum purification period using distilled waster was 1 h instead of the usual range of 1 and 7 days (uses HF and HCl and dialysis). Humic acids could be isolated from tropical peat soils within 7 h (i.e. 4 h extraction, 2 h fractionation, and 1 h purification) instead of the existing period of 2 and 7 days. This could facilitate the idea of producing organic fertilizers such as ammonium-humate and potassium-humate from humified substances since techniques devised in this study did not alter the true nature of the HA. Besides, the technique is rapid, simple

  11. 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.

  12. Method for production of petroselinic acid and OMEGA12 hexadecanoic acid in transgenic plants

    DOEpatents

    Ohlrogge, John B.; Cahoon, Edgar B.; Shanklin, John; Somerville, Christopher R.

    1995-01-01

    The present invention relates to a process for producing lipids containing the fatty acid petroselinic acid in plants. The production of petroselinic acid is accomplished by genetically transforming plants which do not normally accumulate petroselinic acid with a gene for a .omega.12 desaturase from another species which does normally accumulate petroselinic acid.

  13. Method for production of petroselinic acid and OMEGA12 hexadecanoic acid in transgenic plants

    DOEpatents

    Ohlrogge, J.B.; Cahoon, E.B.; Shanklin, J.; Somerville, C.R.

    1995-07-04

    The present invention relates to a process for producing lipids containing the fatty acid, petroselinic acid, in plants. The production of petroselinic acid is accomplished by genetically transforming plants which do not normally accumulate petroselinic acid with a gene for a {omega}12 desaturase from another species which does normally accumulate petroselinic acid. 19 figs.

  14. Evaluation of Molecular Methods for the Detection and Quantification of Pathogen-Derived Nucleic Acids in Sediment

    PubMed Central

    Farkas, Kata; Hassard, Francis; McDonald, James E.; Malham, Shelagh K.; Jones, Davey L.

    2017-01-01

    The accurate detection of pathogens in environmental matrices, such as sediment, is critical in understanding pathogen fate and behavior in the environment. In this study, we assessed the usefulness of methods for the detection and quantification of Vibrio spp. and norovirus (NoV) nucleic acids in sediment. For bacteria, a commonly used direct method using hexadecyltrimethylammonium bromide (CTAB) and phenol-chloroform-isoamyl alcohol (PCI) extraction was optimized, whereas for NoV, direct and indirect (virus elution—concentration) methods were evaluated. For quantification, commercially available quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) kits were tested alongside a digital PCR (dPCR) approach. CTAB-based extraction combined with 16 h polyethylene glycol 6000 (PEG6000) precipitation was found to be suitable for the direct extraction of high abundance bacterial and viral nucleic acids. For the indirect extraction of viral RNA, beef extract-based elution followed by PEG6000 precipitation and extraction using the NucliSENS® MiniMag® Nucleic Acid Purification System and the PowerViral® Environmental RNA/DNA Isolation Kit and qRT-PCR resulted in 83–112 and 63–69% recoveries of NoV, respectively. dPCR resulted in lower viral recoveries (47 and 9%) and ~4 orders of magnitude lower Vibrio concentrations (3.6–4.6 log10 gc/100 g sediment) than was observed using qPCR. The use of internal controls during viral quantification revealed that the RT step was more affected by inhibitors than the amplification. The methods described here are suitable for the enumeration of viral and/or bacterial pathogens in sediment, however the use of internal controls to assess efficiency is recommended. PMID:28174565

  15. Method of Identifying a Base in a Nucleic Acid

    DOEpatents

    Fodor, Stephen P. A.; Lipshutz, Robert J.; Huang, Xiaohua

    1999-01-01

    Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

  16. Exonuclease III-Assisted Target Recycling Amplification Coupled with Liposome-Assisted Amplification: One-Step and Dual-Amplification Strategy for Highly Sensitive Fluorescence Detection of DNA.

    PubMed

    Zhou, Fulin; Li, Baoxin

    2015-07-21

    Detection of ultralow concentration of specific DNA sequence is a central challenge in the early diagnosis of gene-related disease and biodefense application. Herein, we report a dual-amplification strategy for highly sensitive fluorescence detection of DNA. In this proposed strategy, a dumbbell-shaped DNA probe is designed to integrate target binding, magnetic separation, and signal response. In the presence of specific DNA target, the multifunctional dumbbell probe can initiate exonuclease III (Exo III)-aided target recycling amplification, and, in the meantime, generate a large number of fluorescein (FAM)-encapsulated liposomes. The developed method offers very high sensitivity due to primary amplification via numerous FAM from a liposome and secondary amplification via target recycling amplification. The detection limit of the proposed method can reach 4 aM, which is much lower than that of the Exo III-aided target recycling technique applied for DNA quantification without FAM-encapsulated liposomes amplification. Moreover, the dual-signal amplification process can be completed one-step in this system. Therefore, this method provides a simple, isothermal, and low-cost approach for sensitive detection of DNA and holds a great potential for early diagnosis in gene-related diseases.

  17. Evaluation of Two Loop-mediated Isothermal Amplification Methods for the Detection of Salmonella Enteritidis and Listeria Monocytogenes in Artificially Contaminated Ready-to-Eat Fresh Produce

    PubMed Central

    Birmpa, Angeliki; Kalogeropoulos, Konstantinos; Kokkinos, Petros

    2015-01-01

    In the present study, the effectiveness of two loop-mediated isothermal amplification (LAMP) assays was evaluated. Samples of romaine lettuce, strawberries, cherry tomatoes, green onions and sour berries were inoculated with known dilutions (100-108 CFU/g of produce) of S. Enteritidis and L. monocytogenes. With LAMP, assay pathogens can be detected in less than 60 min. The limits of detection of S. Enteritidis and L. monocytogenes depended on the food sample tested and on the presence of enrichment step. After enrichment steps, all food samples were found positive even at low initial pathogen levels. The developed LAMP, assays, are expected to become a valuable, robust, innovative, powerful, cheap and fast monitoring tool, which can be extensively used for routine analysis, and screening of contaminated foods by the food industry and the Public Food Health Authorities. PMID:27800413

  18. Physical methods of nucleic acid transfer: general concepts and applications

    PubMed Central

    Villemejane, Julien; Mir, Lluis M

    2009-01-01

    Physical methods of gene (and/or drug) transfer need to combine two effects to deliver the therapeutic material into cells. The physical methods must induce reversible alterations in the plasma membrane to allow the direct passage of the molecules of interest into the cell cytosol. They must also bring the nucleic acids in contact with the permeabilized plasma membrane or facilitate access to the inside of the cell. These two effects can be achieved in one or more steps, depending upon the methods employed. In this review, we describe and compare several physical methods: biolistics, jet injection, hydrodynamic injection, ultrasound, magnetic field and electric pulse mediated gene transfer. We describe the physical mechanisms underlying these approaches and discuss the advantages and limitations of each approach as well as its potential application in research or in preclinical and clinical trials. We also provide conclusions, comparisons, and projections for future developments. While some of these methods are already in use in man, some are still under development or are used only within clinical trials for gene transfer. The possibilities offered by these methods are, however, not restricted to the transfer of genes and the complementary uses of these technologies are also discussed. As these methods of gene transfer may bypass some of the side effects linked to viral or biochemical approaches, they may find their place in specific clinical applications in the future. This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009 PMID:19154421

  19. Comparison of real-time PCR, reverse transcriptase real-time PCR, loop-mediated isothermal amplification, and the FDA conventional microbiological method for the detection of Salmonella spp. in produce.

    PubMed

    Zhang, Guodong; Brown, Eric W; González-Escalona, Narjol

    2011-09-01

    Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (10(5) and <10(1) CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities.

  20. DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth

    PubMed Central

    Moncada, Ximena; Payacán, Claudia; Arriaza, Francisco; Lobos, Sergio; Seelenfreund, Daniela; Seelenfreund, Andrea

    2013-01-01

    Background Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. Methodology We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. Conclusions Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials. PMID:23437166

  1. Identification of follicular lymphoma by polymerase chain reaction amplification of the t(14;18) translocation: An improved method for staging of malignancy and detection of minimal residual disease

    SciTech Connect

    La Spada, A.R.; Lakey, C.; Hanke, D.C.

    1994-09-01

    60 to 85% of follicular non-Hodgkin`s lymphomas have a reciprocal translocation between chromosomes 14 and 18 (t(14q21;18q32)). As a result of this translocation, the joining region of the immunoglobulin heavy chain gene (J{sub H}) on chromosome 14 is juxtaposed to the bcl-2 gene on chromosome 18. Polymerase chain reaction (PCR) amplification across the translocation breakpoint may be used to detect follicular lymphoma cells in the peripheral blood and bone marrow, as morphological examination for lymphoma cells is extremely insensitive. We performed PCR assays with a universal J{sub H} primer and pairs of major breakpoint region (MBR) primers or minor cluster region (MCR) primers from the bcl-2 gene. PCR products were then visualized on ethidium-stained agarose gels, along with size markers and appropriate positive and negative controls. We began by comparing the PCR assay to Southern blot analysis and found that Southern blot analysis detected 9 translocations in 24 follicular lymphomas (37.5%), while PCR amplification detected 13 (58.3%), making it the more sensitive technique. By using adjacent MBR and MCR primer pairs in the PCR assay, we were also able to attain very high specificities (>95%). As detection of minimal residual disease after therapy is likely important for the prognosis and further management of these patients, we decided to use our PCR assay to evaluate peripheral blood stem cell preparations from patients undergoing autologous bone marrow transplantation. Of the 26 patients analyzed thus far, 4 (15%) have been found positive for MBR translocations. These results indicate that PCR amplification of the t(14;18) translocation is a sensitive and specific method for detection of follicular lymphoma cells, and therefore has potential application as a tool not only for the staging but also for the management of this malignancy.

  2. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

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

    2003-11-04

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

  3. Optofluidic analysis system for amplification-free, direct detection of Ebola infection

    NASA Astrophysics Data System (ADS)

    Cai, H.; Parks, J. W.; Wall, T. A.; Stott, M. A.; Stambaugh, A.; Alfson, K.; Griffiths, A.; Mathies, R. A.; Carrion, R.; Patterson, J. L.; Hawkins, A. R.; Schmidt, H.

    2015-09-01

    The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.

  4. 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

  5. Method for producing labeled single-stranded nucleic acid probes

    DOEpatents

    Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

    1999-10-19

    Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

  6. Application of culture culture-independent molecular biology based methods to evaluate acetic acid bacteria diversity during vinegar processing.

    PubMed

    Ilabaca, Carolina; Navarrete, Paola; Mardones, Pamela; Romero, Jaime; Mas, Albert

    2008-08-15

    Acetic acid bacteria (AAB) are considered fastidious microorganisms because they are difficult to isolate and cultivate. Different molecular approaches were taken to detect AAB diversity, independently of their capacity to grow in culture media. Those methods were tested in samples that originated during traditional vinegar production. Bacterial diversity was assessed by analysis of 16S rRNA gene, obtained by PCR amplifications of DNA extracted directly from the acetification container. Bacterial composition was analyzed by RFLP-PCR of 16S rRNA gene, Temporal Temperature Gradient Gel Electrophoresis (TTGE) separation of amplicons containing region V3-V5 of 16S rRNA gene and cloning of those amplicons. TTGE bands and clones were grouped based on their electrophoretic pattern similarity and sequenced to be compared with reference strains. The main microorganism identified in vinegar was Acetobacter pasteurianus, which at the end of the acetification process was considered to be the only microorganism present. The diversity was the highest at 2% acetic acid, where indefinite species of Gluconacetobacter xylinus/europaeus/intermedius were also present.

  7. 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.

  8. Compositions and method for controlling precipitation when acidizing sour wells

    SciTech Connect

    Dill, W.R.; Walker, M.L.

    1989-12-19

    This patent describes an acidizing composition for treating a sour well. It comprises: a base acid solution having an initial ph below 1.9; an iron sequestering agent to combine with iron present in the solution comprising at least one compound selected from the group consisting of aminopolycarboxylic acids, hydroxycarboxylic acids, cyclic polyethers and derivatives of the acids and ethers present in an amount of from about 0.25 to about 5 percent by weight of the acid solution; and a sulfide modifier to combine with sulfides present in the solution comprising at least one member selected from the group consisting of an aldehyde, acetal, hemiacetal and any other compound capable of forming an aldehyde in solution, present in an amount of from about 1 to about 4 percent by weight of the acid solution, whereby precipitation of ferric hydroxide, ferrous sulfide and elemental sulfur is inhibited as acid spending occurs.

  9. In situ synthesis carbonated hydroxyapatite layers on enamel slices with acidic amino acids by a novel two-step method.

    PubMed

    Wu, Xiaoguang; Zhao, Xu; Li, Yi; Yang, Tao; Yan, Xiujuan; Wang, Ke

    2015-09-01

    In situ fabrication of carbonated hydroxyapatite (CHA) remineralization layer on an enamel slice was completed in a novel, biomimetic two-step method. First, a CaCO3 layer was synthesized on the surface of demineralized enamel using an acidic amino acid (aspartic acid or glutamate acid) as a soft template. Second, at the same concentration of the acidic amino acid, rod-like carbonated hydroxyapatite was produced with the CaCO3 layer as a sacrificial template and a reactant. The morphology, crystallinity and other physicochemical properties of the crystals were characterized using field emission scanning electron microscopy (FESEM), Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy-dispersive X-ray analysis (EDAX), respectively. Acidic amino acid could promote the uniform deposition of hydroxyapatite with rod-like crystals via absorption of phosphate and carbonate ions from the reaction solution. Moreover, compared with hydroxyapatite crystals coated on the enamel when synthesized by a one-step method, the CaCO3 coating that was synthesized in the first step acted as an active bridge layer and sacrificial template. It played a vital role in orienting the artificial coating layer through the template effect. The results show that the rod-like carbonated hydroxyapatite crystals grow into bundles, which are similar in size and appearance to prisms in human enamel, when using the two-step method with either aspartic acid or acidic glutamate (20.00 mmol/L).

  10. Method of preparing and using and composition for acidizing subterranean formations

    SciTech Connect

    Dill, W.R.

    1984-08-21

    A composition and method of acidizing or fracturing a subterranean formation comprises contacting the formation with a composition comprising an acid, urea, and a selected gelling agent. The urea is present in an amount sufficient to extend the viscous stability of the gelled acid composition in comparison to the acid and gelling agent alone.

  11. Loop-mediated isothermal amplification of single pollen grains.

    PubMed

    Bektaş, Ali; Chapela, Ignacio

    2014-08-01

    The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop-mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.

  12. HPLC method for the simultaneous quantification of the major organic acids in Angeleno plum fruit

    NASA Astrophysics Data System (ADS)

    Wang, Yanwei; Wang, Jing; Cheng, Wei; Zhao, Zhilei; Cao, Jiankang

    2014-08-01

    A method was developed to profile major organic acids in Angeleno fruit by high performance liquid chromatography. Organic acids in plum were extracted by water with ultra- sonication at 50°C for 30 min. The extracts were chromatographed on Waters Atlantis T3 C18 column (4.6 mm×250 mm, 5 μm) with 0.01mol/L sulfuric acid and water as mobile phase, and flow rate was 0.5 ml/min. The column temperature was 40C, and chromatography was monitored by a diode array detector at 210 nm. The result showed that malic acid, citric acid, tartaric acid, oxalic acid, pyruvic acid, acetic acid, succinic acid in Angeleno plum, and the malic acid was the major organic acids. The coefficient of determination of the standard calibration curve is R2 > 0.999. The organic acids recovery ranged from 99.11% for Malic acid to 106.70% for Oxalic acid, and CV (n=6) ranged from 0.95% for Malic acid to 6.23% for Oxalic acid, respectively. The method was accurate, sensitive and feasible in analyzing the organic acids in Angeleno plum.

  13. Molecular cloning of a cysteine proteinase cDNA from adult Ancylostoma ceylanicum by the method of rapid amplification of cDNA ends using polymerase chain reaction.

    PubMed

    Mieszczanek, J; Kofta, W; Wedrychowicz, H

    2000-12-01

    The hookworm Ancylostoma ceylanicum is a parasite of great importance in human and veterinary medicine. The most promising vaccination trials against hookworm infections are based on antigens belonging to the proteinase family. The aim of the present research was to isolate a cysteine proteinase gene from A. ceylanicum. This was achieved by rapid amplification of cDNA ends using polymerase chain reaction (RACE-PCR). A set of consensus oligonucleotide primers was designed to anneal to the conserved coding regions of cysteine proteinase. The PCR products were cloned and sequenced. The novel sequence displayed a high degree of homology with genes of cysteine proteinases known from other hookworm species. In the coding region the nucleotide identity with accp-1, the cysteine proteinase gene of A. caninum, reaches 84.3%. Analysis of the expression of acey-1. the cysteine proteinase gene of A. ceylanicum, suggests that it is produced exclusively in the gland cells of either adult worms or blood-feeding stages of A. ceylanicum.

  14. Development and evaluation of a simple assay for Marburg virus detection using a reverse transcription-loop-mediated isothermal amplification method.

    PubMed

    Kurosaki, Yohei; Grolla, Allen; Fukuma, Aiko; Feldmann, Heinz; Yasuda, Jiro

    2010-07-01

    Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 10(2) copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 10(4) 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic.

  15. A rapid loop-mediated isothermal amplification (LAMP) method for detection of the macrolide-streptogramin type B resistance gene msrA in Staphylococcus aureus.

    PubMed

    Mu, Xiao-Qin; Liu, Bin-Bin; Hui, Ephraim; Huang, William; Yao, Li-Chen; Duo, Li-Bo; Sun, Wen-Ying; Li, Gui-Qiu; Wang, Fu-Xiang; Liu, Shu-Lin

    2016-12-01

    Macrolide-streptogramin type B resistance (the MSB phenotype) is a multidrug resistance phenotype in Staphylococcus aureus conferred by the resistance gene msrA. However, bacteria having the MSB phenotype are susceptible to lincosamides and 16-membered ring macrolides, which makes profiling resistance genes necessary and urgent for timely and appropriate use of antimicrobials. In this study, the loop-mediated isothermal amplification (LAMP) assay was optimized for prompt detection of the msrA gene. msrA gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database and primers were designed using the LAMP primer designing software PrimerExplorer v.4, which together recognize seven distinct regions of the msrA gene. The specific LAMP primer set designed in this study could amplify the msrA gene within 25min at an isothermal temperature of 62°C. More importantly, the msrA gene could be detected at a sensitivity as low as 100pg. Furthermore, this optimized LAMP assay provided swift detection of the msrA gene even directly from human specimens. In conclusion, this assay may have great clinical application potential for detection of the msrA gene.

  16. A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7.

    PubMed

    Ravan, Hadi; Amandadi, Mojdeh; Sanadgol, Nima

    2016-02-01

    E. coli O157:H7 is one of the most important foodborne pathogen that causes some human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six special primers that target a highly specific 299-bp region of the Z3276 gene for the detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive results were only obtained from E. coli O157:H7 strains. The sensitivity level of the Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial culture. Moreover, the LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 10(3) CFU/mL without pre-enrichment and 10 CFU/mL after a 4-h pre-enrichment. In conclusion, the present LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific diagnosis of E. coli O157:H7 strains in resource limited laboratories.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. Universal nucleic acids sample preparation method for cells, spores and their mixture

    DOEpatents

    Bavykin, Sergei [Darien, IL

    2011-01-18

    The present invention relates to a method for extracting nucleic acids from biological samples. More specifically the invention relates to a universal method for extracting nucleic acids from unidentified biological samples. An advantage of the presently invented method is its ability to effectively and efficiently extract nucleic acids from a variety of different cell types including but not limited to prokaryotic or eukaryotic cells and/or recalcitrant organisms (i.e. spores). Unlike prior art methods which are focused on extracting nucleic acids from vegetative cell or spores, the present invention effectively extracts nucleic acids from spores, multiple cell types or mixtures thereof using a single method. Important that the invented method has demonstrated an ability to extract nucleic acids from spores and vegetative bacterial cells with similar levels effectiveness. The invented method employs a multi-step protocol which erodes the cell structure of the biological sample, isolates, labels, fragments nucleic acids and purifies labeled samples from the excess of dye.

  2. Kinetic Hairpin Oligonucleotide Blockers for Selective Amplification of Rare Mutations

    PubMed Central

    Jia, Yanwei; Sanchez, J. Aquiles; Wangh, Lawrence J.

    2014-01-01

    Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals. PMID:25082368

  3. Fast high-throughput method for the determination of acidity constants by capillary electrophoresis: I. Monoprotic weak acids and bases.

    PubMed

    Fuguet, Elisabet; Ràfols, Clara; Bosch, Elisabeth; Rosés, Martí

    2009-04-24

    A new and fast method to determine acidity constants of monoprotic weak acids and bases by capillary zone electrophoresis based on the use of an internal standard (compound of similar nature and acidity constant as the analyte) has been developed. This method requires only two electrophoretic runs for the determination of an acidity constant: a first one at a pH where both analyte and internal standard are totally ionized, and a second one at another pH where both are partially ionized. Furthermore, the method is not pH dependent, so an accurate measure of the pH of the buffer solutions is not needed. The acidity constants of several phenols and amines have been measured using internal standards of known pK(a), obtaining a mean deviation of 0.05 pH units compared to the literature values.

  4. Separating acetic acid from furol (furfural) by electrodialysis method

    SciTech Connect

    Guan, S.F.; Li, C.S. Ye, S.T.; Shen, S.Y.; Wang, Y.T.; Yu, S.H.

    1981-01-01

    Furfural production by hydrolysis of fibrous plant materials is accompanied by formation of acetic acid in amounts depending on the material used. The amount of acetic formed in the hydrolysis of the fruit shell of oil-tea camellia (Camellia oleosa) (an oilseed-bearing tree) is equal to the amount of furfural. The acetic acid can be separated from the furfural and concentrated to 10% by electrodialysis. A smaller amount of furfural is separated with acetic acid.

  5. Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.

    2002-01-01

    A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

  6. Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.

    2003-12-09

    A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

  7. Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples.

    PubMed

    Cowell, Annie N; Loy, Dorothy E; Sundararaman, Sesh A; Valdivia, Hugo; Fisch, Kathleen; Lescano, Andres G; Baldeviano, G Christian; Durand, Salomon; Gerbasi, Vince; Sutherland, Colin J; Nolder, Debbie; Vinetz, Joseph M; Hahn, Beatrice H; Winzeler, Elizabeth A

    2017-02-07

    Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens.

  8. Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

    PubMed Central

    Loy, Dorothy E.; Sundararaman, Sesh A.; Valdivia, Hugo; Fisch, Kathleen; Lescano, Andres G.; Baldeviano, G. Christian; Durand, Salomon; Gerbasi, Vince; Sutherland, Colin J.; Nolder, Debbie; Vinetz, Joseph M.; Hahn, Beatrice H.

    2017-01-01

    ABSTRACT Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. PMID:28174312

  9. 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.

  10. Rapid detection of Brucella spp. using loop-mediated isothermal amplification (LAMP).

    PubMed

    Chen, Shouyi; Li, Xunde; Li, Juntao; Atwill, Edward R

    2013-01-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Livestock that are most vulnerable to brucellosis include cattle, goats, and pigs. Brucella spp. cause serious health problems to humans and animals and economic losses to the livestock industry. Traditional methods for detection of Brucella spp. take 48-72 h (Kumar et al., J Commun Dis 29:131-137, 1997; Barrouin-Melo et al., Res Vet Sci 83:340-346, 2007) that do not meet the food industry's need of rapid detection. Therefore, there is an urgent need of fast, specific, sensitive, and inexpensive method for diagnosing of Brucella spp. Loop-mediated isothermal amplification (LAMP) is a method to amplify nucleic acid at constant temperatures. Amplification can be detected by visual detection, fluorescent stain, turbidity, and electrophoresis. We targeted at the Brucella-specific gene omp25 and designed LAMP primers for detection of Brucella spp. Amplification of DNA with Bst DNA polymerase can be completed at 65 °C in 60 min. Amplified products can be detected by SYBR Green I stain and 2.0% agarose gel electrophoresis. The LAMP method is feasible for detection of Brucella spp. from blood and milk samples.

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

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2000-01-01

    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.

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

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2004-10-12

    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.

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

    SciTech Connect

    Antwerp, W.P. van; Mastrototaro, J.J.

    2000-01-04

    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.

  14. A Novel Method for Presenting the Amino Acids in an Introductory Biochemistry Course.

    ERIC Educational Resources Information Center

    Kuehl, LeRoy

    1978-01-01

    Introduces an approach to teaching amino acids that employs the use of a poem containing information on the structure and properties of amino acids, and of slides illustrating the poem. Student response to the method was positive. (MA)

  15. An investigation of PCR inhibition using Plexor(®) -based quantitative PCR and short tandem repeat amplification.

    PubMed

    Thompson, Robyn E; Duncan, George; McCord, Bruce R

    2014-11-01

    A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor(®) real-time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and CT takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex(®) 16 HS System. The overall results demonstrate that real-time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.

  16. Method for removing acid gases from a gaseous stream

    DOEpatents

    Gorin, Everett; Zielke, Clyde W.

    1981-01-01

    In a process for hydrocracking a heavy aromatic polynuclear carbonaceous feedstock containing reactive alkaline constituents to produce liquid hydrocarbon fuels boiling below about 475.degree. C. at atmospheric pressure by contacting the feedstock with hydrogen in the presence of a molten metal halide catalyst, thereafter separating a gaseous stream containing hydrogen, at least a portion of the hydrocarbon fuels and acid gases from the molten metal halide and regenerating the molten metal halide, thereby producing a purified molten metal halide stream for recycle to the hydrocracking zone, an improvement comprising; contacting the gaseous acid gas, hydrogen and hydrocarbon fuels-containing stream with the feedstock containing reactive alkaline constituents to remove acid gases from the acid gas containing stream. Optionally at least a portion of the hydrocarbon fuels are separated from gaseous stream containing hydrogen, hydrocarbon fuels and acid gases prior to contacting the gaseous stream with the feedstock.

  17. Energy recovery method and system for combined sulphuric acid and phosphoric acid manufacturing plant

    SciTech Connect

    Cameron, G. M.; Orlando, J. V.

    1985-01-22

    In conventional processes for manufacturing phosphoric acid, sulphuric acid is reacted with phosphate rock to produce weak phosphoric acid which is concentrated using steam from the associated sulphuric acid manufacturing operation. Low grade heat from the absorbers and drier of the sulphuric acid manufacturing plant has been wasted. According to the invention waste heat from the drier and one or more absorbers of the sulphuric acid manufacturing plant is used in all the evaporators of the phosphoric acid plant. The evaporators all operate at low pressures and their heaters are arranged in series, to enable the heat to be used at the relatively low temperatures available. The valuable steam is thus freed for other uses.

  18. Loop-Mediated Isothermal Amplification (LAMP) for Detection of Phytoplasmas in the Field.

    PubMed

    Dickinson, Matt

    2015-01-01

    Loop-mediated isothermal amplification (LAMP) is a nucleic acid-based detection method with many applications. This chapter details its use for detection of phytoplasma diseases, combining a rapid 2-min DNA extraction method with real-time LAMP product detection such that the entire procedure can be undertaken and completed in the field within 40 min. Furthermore, the assays include an anneal curve validation step to guard against false positives and an assay for host plant DNA to guard against false negatives.

  19. A general solution for opening double-stranded DNA for isothermal amplification

    PubMed Central

    Chen, Gangyi; Dong, Juan; Yuan, Yi; Li, Na; Huang, Xin; Cui, Xin; Tang, Zhuo

    2016-01-01

    Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites. Then, the nucleoprotein can invade the double-stranded template to form heteroduplex in the presence of ATP, resulting in the strand exchange. The ATP regeneration system could be eliminated by using high concentration of ATP, and the 3′-OH terminal of the invasion strand can be recognized by other DNA modifying enzymes such as DNA polymerase or DNA ligase. Moreover, dATP was found to be a better cofactor for RecA, which make the system more compatible to DNA polymerase. The method described here is a general solution to open dsDNA, serving as a platform to develop more isothermal nucleic acids detection methods for real DNA samples based on it. PMID:27687498

  20. Chromogenic in situ hybridization to detect HER-2/neu gene amplification in histological and ThinPrep-processed breast cancer fine-needle aspirates: a sensitive and practical method in the trastuzumab era.

    PubMed

    Vocaturo, Amina; Novelli, Flavia; Benevolo, Maria; Piperno, Giulia; Marandino, Ferdinando; Cianciulli, Anna Maria; Merola, Roberta; Donnorso, Raffaele Perrone; Sperduti, Isabella; Buglioni, Simonetta; Mottolese, Marcella

    2006-09-01

    The increasing evidence of trastuzumab efficacy in breast cancer (BC) patients means that an accurate and reproducible evaluation of HER-2 statusis of paramount importance in histological and in cytological samples. Currently, the two main methods used to analyze HER-2 amplification or overexpression are fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Although the two methods are strongly correlated for histological tissue, the evaluation of tumor morphology through FISH may be difficult and fluorescence fades quickly. These limitations can be overcome by chromogenic in situ hybridization (CISH), which can visualize the amplification product along with morphological features. In view of this, in the present study, we analyzed the usefulness of CISH on formalin-fixed, paraffin-embedded (FFPE) BC specimens and investigated whether CISH can be a valid technique in the determination of HER-2 status for fine-needle aspirates (FNAs) processed by liquid-based cytology. The results we obtained in a retrospective series of 111 FFPE BC specimens demonstrated good concordance between CISH and IHC and between CISH and FISH. The former concordance was comparable with that observed between FISH and IHC. When CISH was applied to a prospective series of 53 FNAs, from surgically removed BC, our data showed evidence of a higher concordance of results between liquid-based cytology and the companion FFPE tissues using CISH rather than HercepTesttrade mark. Therefore, CISH analysis, which is avaluable and reproducible alternative to FISH for selecting breast cancer patients for trastuzumab therapy, can lower false-positive immunocytochemistry findings in ThinPrep-processed FNAs.

  1. Gene and library synthesis without amplification: polymerase step reaction (PSR).

    PubMed

    Lee, Zhuo-Bin; Firnhaber, Christopher; Clarke, Jesse; DeDecker, Brian S

    2015-09-01

    Current gene synthesis methods often incorporate a PCR amplification step in order to yield final material sufficient for resolution from multiple off-products. These amplification steps can cause stochastic sampling effects that propagate errors in gene synthesis or decrease variability when applied to the construction of randomized libraries. We have developed a simple DNA polymerase-based gene synthesis reaction, polymerase step reaction (PSR), that assembles DNA oligonucleotides in a unidirectional fashion without the need for amplification. We demonstrate that PSR is efficient, with little off-product production, no detectable error propagation, and maximized variability in the synthesis of a phage display library.

  2. Preparation of DNA-containing extract for PCR amplification

    DOEpatents

    Dunbar, John M.; Kuske, Cheryl R.

    2006-07-11

    Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

  3. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  4. Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.

    PubMed

    Su, Xin; Li, Zehao; Yan, Xinzhong; Wang, Lei; Zhou, Xu; Wei, Lin; Xiao, Lehui; Yu, Changyuan

    2017-03-21

    Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.

  5. Method for producing 3-hydroxypropionic acid and other products

    DOEpatents

    Lynch, Michael D.; Gill, Ryan T.; Lipscomb, Tanya E.W.

    2016-08-30

    This invention relates to metabolically engineered microorganism strains, such as bacterial strains, in which there is an increased utilization of malonyl-CoA for production of a chemical product, which includes 3-hydroxypropionic acid.

  6. Methods for producing 3-hydroxypropionic acid and other products

    DOEpatents

    Lynch, Michael D.; Gill, Ryan T.; Lipscomb, Tanya E. W.

    2016-07-12

    This invention relates to metabolically engineered microorganism strains, such as bacterial strains, in which there is an increased utilization of malonyl-CoA for production of a chemical product, which includes 3-hydroxypropionic acid.

  7. A simple and cost-effective method for preparing DNA from the hard tooth tissue, and its use in polymerase chain reaction amplification of amelogenin gene segment for sex determination in an Indian population.

    PubMed

    Sivagami, A V; Rao, A R; Varshney, U

    2000-05-15

    The use of teeth as an important resource in the analysis of forensic case history by polymerase chain reaction (PCR) or other related methods has been reported. However, a major drawback in using teeth has been that the DNA is present only in trace amounts, and the methods to recover DNA from the flinty material have not been efficient or cost effective. In this report, we describe a method to prepare DNA from the hard tooth tissues. Our studies show that ultrasonication of teeth samples yields sufficient amounts of good quality DNA useful for PCR-based diagnostic methods. The teeth could serve as a reliable source of DNA for amplification-based forensic methods in sex determination. DNA could be obtained from any tooth, regardless of the age of subject. Furthermore, by using the AMEL gene-based primers in PCR, we have shown that the AMEL gene serves as a good marker for sex determination in the Indian population. In our study, the PCR-based method was sensitive and proved to be successful for sex determination with a complete specificity.

  8. Materials and methods for efficient lactic acid production

    DOEpatents

    Zhou, Shengde; Ingram, Lonnie O'Neal; Shanmugam, Keelnatham T.; Yomano, Lorraine; Grabar, Tammy B.; Moore, Jonathan C.

    2009-12-08

    The present invention provides derivatives of ethanologenic Escherichia coli K011 constructed for the production of lactic acid. The transformed E. coli of the invention are prepared by deleting the genes that encode competing pathways followed by a growth-based selection for mutants with improved performance. These transformed E. coli are useful for providing an increased supply of lactic acid for use in food and industrial applications.

  9. Materials and methods for efficient lactic acid production

    SciTech Connect

    Zhou, Shengde; Ingram, Lonnie O'Neal; Shanmugam, Keelnatham T; Yomano, Lorraine; Grabar, Tammy B; Moore, Jonathan C

    2013-04-23

    The present invention provides derivatives of Escherichia coli constructed for the production of lactic acid. The transformed E. coli of the invention are prepared by deleting the genes that encode competing pathways followed by a growth-based selection for mutants with improved performance. These transformed E. coli are useful for providing an increased supply of lactic acid for use in food and industrial applications.

  10. Comparative Oxidative Stability of Fatty Acid Alkyl Esters by Accelerated Methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several fatty acid alkyl esters were subjected to accelerated methods of oxidation, including EN 14112 (Rancimat method) and pressurized differential scanning calorimetry (PDSC). Structural trends elucidated from both methods that improved oxidative stability included decreasing the number of doubl...

  11. Validation of a multi-analyte HPLC-DAD method for determination of uric acid, creatinine, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid and 2-methylhippuric acid in human urine.

    PubMed

    Remane, Daniela; Grunwald, Soeren; Hoeke, Henrike; Mueller, Andrea; Roeder, Stefan; von Bergen, Martin; Wissenbach, Dirk K

    2015-08-15

    During the last decades exposure sciences and epidemiological studies attracts more attention to unravel the mechanisms for the development of chronic diseases. According to this an existing HPLC-DAD method for determination of creatinine in urine samples was expended for seven analytes and validated. Creatinine, uric acid, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid, and 2-methylhippuric acid were separated by gradient elution (formate buffer/methanol) using an Eclipse Plus C18 Rapid Resolution column (4.6mm×100mm). No interfering signals were detected in mobile phase. After injection of blank urine samples signals for the endogenous compounds but no interferences were detected. All analytes were linear in the selected calibration range and a non weighted calibration model was chosen. Bias, intra-day and inter-day precision for all analytes were below 20% for quality control (QC) low and below 10% for QC medium and high. The limits of quantification in mobile phase were in line with reported reference values but had to be adjusted in urine for homovanillic acid (45mg/L), niacinamide 58.5(mg/L), and indole-3-acetic acid (63mg/L). Comparison of creatinine data obtained by the existing method with those of the developed method showing differences from -120mg/L to +110mg/L with a mean of differences of 29.0mg/L for 50 authentic urine samples. Analyzing 50 authentic urine samples, uric acid, creatinine, hippuric acid, and 2-methylhippuric acid were detected in (nearly) all samples. However, homovanillic acid was detected in 40%, niacinamide in 4% and indole-3-acetic acid was never detected within the selected samples.

  12. Comparison of automated nucleic acid extraction methods for the detection of cytomegalovirus DNA in fluids and tissues

    PubMed Central

    Waggoner, Jesse J.

    2014-01-01

    Testing for cytomegalovirus (CMV) DNA is increasingly being used for specimen types other than plasma or whole blood. However, few studies have investigated the performance of different nucleic acid extraction protocols in such specimens. In this study, CMV extraction using the Cell-free 1000 and Pathogen Complex 400 protocols on the QIAsymphony Sample Processing (SP) system were compared using bronchoalveolar lavage fluid (BAL), tissue samples, and urine. The QIAsymphonyAssay Set-up (AS) system was used to assemble reactions using artus CMV PCR reagents and amplification was carried out on the Rotor-Gene Q. Samples from 93 patients previously tested for CMV DNA and negative samples spiked with CMV AD-169 were used to evaluate assay performance. The Pathogen Complex 400 protocol yielded the following results: BAL, sensitivity 100% (33/33), specificity 87% (20/23); tissue, sensitivity 100% (25/25), specificity 100% (20/20); urine, sensitivity 100% (21/21), specificity 100% (20/20). Cell-free 1000 extraction gave comparable results for BAL and tissue, however, for urine, the sensitivity was 86% (18/21) and specimen quantitation was inaccurate. Comparative studies of different extraction protocols and DNA detection methods in body fluids and tissues are needed, as assays optimized for blood or plasma will not necessarily perform well on other specimen types. PMID:24765569

  13. Remedial methods for intergranular attack of alloy 600 tubing. Volume 3. Boric acid and acetic acid remedial methods. Final report

    SciTech Connect

    Rubright, M.M.

    1986-06-01

    An important cause of recent tube degradation in recirculating pressurized water reactor (PWR) steam generators with open tube/tubesheet crevices is intergranular attack (IGA) of alloy 600 tubing in the crevice region. The attack appears to occur on the hot leg tubing because of high concentrations of caustic species formed from remnants of past phosphate water treatment, combined with materials from inleakage from freshwater-cooled condensers. The concept of using neutralizers to modify the aggressiveness of the crevice environment was examined. It appears that this can be accomplished by neutralizing the caustic species with an acid. Two ways to apply the acid are by off-line flushing during plant shutdown and by on-line treatment during operation. The substance that appears to be most suitable for off-line flushing is acetic acid, with boric acid as a second choice. Concentrations should be in the range of from 1000 to 5000 ppM. The addition of 1000 to 5000 ppM of a non-ionic detergent in the flush solution should improve penetration of the crevice. Use of preflush lancing to remove sludge on the tubesheet will also help by reducing acid consumption. The requirements for materials to be used in on-line treatment are more stringent because of possible interaction with other components in the secondry system. Boric acid is the only substance that has operational experience. A series of tests are proposed to investigate the behavior of acetic acid and boric acid on tubesheet sludge, on tubesheet/support plate material, and on alloy 600/tubesheet couples. Similarly, areas of uncertainty of on-line treatment with boric acid are its effect on tubesheet/support plate materials and on the rest of the secondary system. 23 refs.

  14. 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.

  15. Amplification of postwildfire peak flow by debris

    USGS Publications Warehouse

    Kean, Jason W.; Mcguire, Luke; Rengers, Francis; Smith, Joel B.; Staley, Dennis M.

    2016-01-01

    In burned steeplands, the peak depth and discharge of postwildfire runoff can substantially increase from the addition of debris. Yet methods to estimate the increase over water flow are lacking. We quantified the potential amplification of peak stage and discharge using video observations of postwildfire runoff, compiled data on postwildfire peak flow (Qp), and a physically based model. Comparison of flood and debris flow data with similar distributions in drainage area (A) and rainfall intensity (I) showed that the median runoff coefficient (C = Qp/AI) of debris flows is 50 times greater than that of floods. The striking increase in Qp can be explained using a fully predictive model that describes the additional flow resistance caused by the emergence of coarse-grained surge fronts. The model provides estimates of the amplification of peak depth, discharge, and shear stress needed for assessing postwildfire hazards and constraining models of bedrock incision.

  16. Amplification of postwildfire peak flow by debris

    NASA Astrophysics Data System (ADS)

    Kean, J. W.; McGuire, L. A.; Rengers, F. K.; Smith, J. B.; Staley, D. M.

    2016-08-01

    In burned steeplands, the peak depth and discharge of postwildfire runoff can substantially increase from the addition of debris. Yet methods to estimate the increase over water flow are lacking. We quantified the potential amplification of peak stage and discharge using video observations of postwildfire runoff, compiled data on postwildfire peak flow (Qp), and a physically based model. Comparison of flood and debris flow data with similar distributions in drainage area (A) and rainfall intensity (I) showed that the median runoff coefficient (C = Qp/AI) of debris flows is 50 times greater than that of floods. The striking increase in Qp can be explained using a fully predictive model that describes the additional flow resistance caused by the emergence of coarse-grained surge fronts. The model provides estimates of the amplification of peak depth, discharge, and shear stress needed for assessing postwildfire hazards and constraining models of bedrock incision.

  17. Method for promoting specific alignment of short oligonucleotides on nucleic acids

    DOEpatents

    Studier, F. William; Kieleczawa, Jan; Dunn, John J.

    1996-01-01

    Disclosed is a method for promoting specific alignment of short oligonucleotides on a nucleic acid polymer. The nucleic acid polymer is incubated in a solution containing a single-stranded DNA-binding protein and a plurality of oligonucleotides which are perfectly complementary to distinct but adjacent regions of a predetermined contiguous nucleotide sequence in the nucleic acid polymer. The plurality of oligonucleotides anneal to the nucleic acid polymer to form a contiguous region of double stranded nucleic acid. Specific application of the methods disclosed include priming DNA synthesis and template-directed ligation.

  18. From Interface to Solution: Integrating Immunoassay with Netlike Rolling Circle Amplification for Ultrasensitive Detection of Tumor Biomarker

    PubMed Central

    Feng, Chang; Bo, Bing; Mao, Xiaoxia; Shi, Hai; Zhu, Xiaoli; Li, Genxi

    2017-01-01

    Both the 3D solution and the 2D interface play important roles in bioanalysis. For the former, reactions can be carried out adequately; while for the latter, interfering substance can be eliminated simply through wash. It is a challenge to integrate the advantages of solution-based assays and the interface-based assays. Here, we report an immuno-NRCA (netlike rolling circle amplification) strategy, which integrates immunoassay with NRCA for the ultrasensitive detection of tumor biomarker, by taking the assay of a tumour marker as an example. In this strategy, immunoreactions occur on interface, while the target-induced signal amplification can be completed totally in solution. As a result, this system has the merits of both solution- and interface-based assays. The whole procedure of this novel strategy is similar to the conventional ELISA, inheriting the usability. But in comparison with ELISA, the performance is greatly improved. The detection limit can be lowered to 5.5 fg/L, making it possible to detect the target tumour marker in one drop of blood. Also, in comparison with established immuno-PCR method, which integrates immunoassay with the commonly used nucleic acid amplification approach, this system has no requirement for thermal cycler owing to the isothermal amplification, and it has the ability to retain the immunoreactivities. So, the new immunoassay method proposed in this study may have more feasible applications in the future. PMID:28042314

  19. Potassium hydroxide-ethylene diamine tetraacetic acid method for the rapid preparation of small-scale PCR template DNA from actinobacteria.

    PubMed

    Sun, Zhibin; Huang, Yan; Wang, Yanzhuo; Zhao, Yuguo; Cui, Zhongli

    2014-01-01

    Genomic DNA extraction from Gram-positive bacteria is a laborious and time-consuming process. A rapid and convenient method was established to extract genomic DNA from a single colony as a PCR template. KOH-EDTA is used as a lysis buffer to disrupt the cell envelope, releasing genomic DNA, and Tris-HCl (pH = 4) is then added to neutralize the lysate. The lysate can be used directly as a template for PCR amplification. 16S rDNA was successfully amplified from Gram-positive bacteria from the genera of Bacillus, Streptomyces, Micromonospora, Nonomuraea, Microbispora, and Staphylococcus. Amplification of the trpB gene indicated that this method could also be applied to the amplification of functional genes. Compared to colony PCR methods without KOH-EDTA, this method is extremely fast and efficient, and it is applicable to high-throughput PCR amplifications.

  20. 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

  1. Simultaneous multiple target detection in real-time loop-mediated isothermal amplification.

    PubMed

    Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C

    2012-08-01

    Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 1-4 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.

  2. 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.

  3. Determination of aqueous acid-dissociation constants of aspartic acid using PCM/DFT method

    NASA Astrophysics Data System (ADS)

    Sang-Aroon, Wichien; Ruangpornvisuti, Vithaya

    Determination of acid-dissociation constants, pKa, of aspartic acid in aqueous solution, using density functional theory calculations combined with the conductor-like polarizable continuum model (CPCM) and with integral-equation-formalism polarizable continuum model (IEFPCM) based on the UAKS and UAHF radii, was carried out. The computed pKa values derived from the CPCM and IEFPCM with UAKS cavity model of bare structures of the B3LYP/6-31+G(d,p)-optimized tetrahydrated structures of aspartic acid species are mostly close to the experimental pKa values.0

  4. 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