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Pre-menstrual Migraine!!!!
I am 41, had my only child at 22, and have not had any sort of chemotherapy,...
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I am 41, had my only child at 22, and have not had any sort of chemotherapy, hysterectomy, or radiation. For the last six years (since I turned 35) I have had pre-menstrual migraines that last about two days. Up until last summer they were mostly the intense pain in my temples and nausea but then they changed to those symptoms, plus vomiting: 3 or 4x over those two days each month. I vomit even if there is nothing in my stomach! I've tried acupuncture, birth control pills, estrogen patch, NOTHING works! I have Sumatriptan but it doesn't always work and I really hate to take an Rx pill, not to mention it has a weird side effect that makes it feel like my throat is closing up.
Does anyone else have this kind of migraine? I am at my wits' end. I hate when my period approaches because I feel like it's a death sentence; i don't know what the solution is and I can't go on like this. It's no way to live. If someone out there has found a way to cope without medicating yourself to the gills, please let me know because I can't take much more. Thanks and God Bless.
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Pre-menstrual Migraine!!!!
I am 41, had my only child at 22, and have not had any sort of chemotherapy,...
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Haizhou Hotel Haining
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Tea and The Amazing Race!
... to try our luck with a bus. But time was ticking and no bus was in sight. After walking a half mile to an intersection where we thought our chances at hailing a taxi would improve, we learned, sadly, that was not the case. The next thing we knew, though, Jordan was talking to a man in a small sedan with a young boy in the back seat. A few words were exchanged and Jordan yelled to Alison and me, 'Get in. Get in. He'll drive you.' Craig joined ...
Haggling and Hangzhou
... Hangzhou. Craig, Alison and I are the oldies here among young people from all over the world. This is a first for me. Alison dethroned Jordan as the Hearts champion tonight as we planned our day for Saturday. Rain is the forecast for tomorrow. Thought of the day: If you don't think men have it easier in the world than women, you've never used a toilet on a train in ...
Last Day of Freedom!!!!
... look like a tiny village. That's how many people there are. After some (more like a lot) of walking, we arrived at the world famous Westlake.
The first thing you notice about Westlake, is the atmosphere. It is so peaceful. You kinda forget that you are even in a city with over 8.7 million residents. People young and old are relaxing and becoming entranced by the sheer beauty of this place. Everyone walks a little ...
Day Trip to Hangzhou to see Cynthia Franklin
I haven't seen the chair of my dissertation, mentor and friend, Cynthia Franklin in more than 12 years and we happened upon each other in China of all places. She was travelling through the country conducting trainings. We were lucky enough to meet up in the beautiful Hangzhou - a short train ride from Shanghai. The lotuses were in bloom so we were treated to a feast for the eyes and soul. It was a beautiful day and so wonderful to catch up with an old friend and ...
Hmm...that was a lot of driving
... architecture, if that's what turns your crank. I repeated my Big Mac meal experience of a few days prior and then we went off to the highlight, an excursion to the silk factory. It's actually really interesting to see the machines unspooling the silk fibres that make up the cacoons of the dead... They kill them :( ...silk worms. Each cacoon produces 1 kilometre of fibre and it takes 8 fibres to create the required diameter of silk thread. ...

subset: commoncrawl


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"I know he will continue to be a great ambassador for our sport and I'm sure Dario has a lot more to contribute to racing, it will just be in a different way than driving an IndyCar," Castroneves said.
Foyt, the only driver with more IndyCar titles than Franchitti with seven, said: "If the doctors tell him he should quit then he should listen to them. I'm the opposite and never would listen, but I probably would have been a lot better off if I did. But Dario's won a lot of races and championships so he has a lot to be proud of. My hat's off to him."
Franchitti could find a role with the Ganassi organization, or perhaps land in the television booth. Andretti suggested Franchitti would be a good fit as a consultant to a manufacturer.
For now, everyone was still trying to adjust to the idea of racing without Franchitti on the track.
"You pinch yourself everyday as firsthand you are witness to the talent, ability, work ethic and start-versus-win ratio of Dario Franchitti," Ganassi team manager Mike Hull said. "Having Dario represent all of us is what's right about motorsports. His representation of the integrity of a true champion is what sets him apart."
AP Sports Writers Mark Long and Jim Vertuno contributed to this report.


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Amy Freedman, wtop.com
WASHINGTON – VDOT will close the left HOV exit from I-66 eastbound to the Outer Loop of the beltway on Friday, June 15 at 10 a.m.
This closure will remain in effect until later this year when it will reopen as a combined HOV-3 and 495 express lanes ramp.
HOV drivers will be directed to use the right lane ramp to the Outer Loop while the closure is in effect. I-66 eastbound HOV traffic on the Inner Loop heading towards Tysons will not be affected.
This work is part of the 495 Express lanes project.
Follow WTOP Traffic and WTOP on Twitter.
(Copyright 2012 by WTOP. All Rights Reserved.)
In Maryland, all sorts of marine life is living among more than 100 sunken ships.


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SIP Trunk Providers India
This page is a list of SIP trunk providers in India. Please keep this list in alphabetical order. India SIP providers looking to add their services can do so in the list below.
- ALTOTELECOM - AltoTelecom is VoIP company that provides SIP Trunking services for Call Centers, hotels, small and large business using a PBX or an Asterisk based predictive dialer such as Vicidial or Goautodial for telemarketing sales, rates under 1 cent per minute to USA, Canada and UK www.altotelecom.com
- Incorpus TeleNetworks Your one stop solution to all voip needs, Call termination, resellers, DIDs, Softswitches, Tollfree, residentail voip, Call center voip.
- AVOXI AVOXI Virtual Call Center Solutions - VoIP Service Provider, provide virtual call center products like SIP trunking and VoIP gateway solutions, with international toll-free numbers. Contact Number 1-800-462-8694.
- CallForwarding - Be present anywhere in the world with toll free forwarding services from CallForwarding.com. Contact Number: 800-231-9802
- i7 Solutions - VOIP Wholesale and Retail i7 Solutions is looking for VoIP Resellers in markets all around the world. We provide the Dialers and Rates your customers want, and the tools for you to control all aspects of your business. i7 Solutions is a one of the very few global VoIP solution providers with direct contracts with local incumbent telecommunications operations across the globe. Our primary business includes A2Z termination services, retails & reseller solutions for VoIP services and also providing calling cards. India TATA CLI is our premium route. Please contact us @ Gtalk and Email ID email@example.com
- UtterU UtterU is leading online voice calling business service provider. UtterU provide Wholesale option for reseller to start international calling business around the world. UtterU also provide SIP trunk facility for all business to making call over the internet at very low cost.
- http://www.doorVaani.com DoorVaani.com is a Business, Residential and Personal VOIP services provider offering VOIP Call Minutes to destinations worldwide, Local Phone Numbers or DIDs in about 60 countries and Toll-free numbers in 5 countries. DoorVaani.com is also an automated web application where ordering of services, making payments and provisioning is done all in real time with no wait time or human interaction needed. Online payments are accepted in 25 currencies including Indian Rupees (INR). Offline payments or Bank Transfers accepted in INR and USD.
- Matrix ComSec - Leading SIP provider in India
- Oxy Communications - India SIP Trunking, Wholesale VoIP, VoIP Origination, VoIP International A-Z, VoIP Billing
- Switch2Voip SIP Trunk provider for business - Switch2Voip a USA based VoIP provider, provides VoIP services for business using a PBX or an Asterisk based predictive dialer such as Vicidial or Goautodial 0.008 per minute to USA and UK www.switch2voip.us. Unlimited channels with every account with no per channel cost, only pay for the minutes. Free USA or Canada DID with every account.
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Publications (27)12.91 Total impact
Article: Application of poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] for splitting of single-walled carbon nanotube bundles in polystyrene/SWCNT composite[Show abstract] [Hide abstract]
ABSTRACT: Dispersions of single-walled carbon nanotubes (SWCNTs) in organic solutions containing poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) were studied by Raman spectroscopy, UV-vis-NIR spectroscopy, and electron microscopy. This polymer interacts with the nanotube resulting in the appearance of a new red-shifted absorption band in the electronic spectrum. This indicates the formation of a charge-transfer complex between MEH-PPV and SWCNTs. Additives of MEH-PPV make it possible to achieve stable suspensions of nanotubes in styrene. A polystyrene/SWCNT/MEH-PPV composite with a high degree of bundle splitting was obtained by polymerization. It was shown that the luminescence intensity of the nanotubes in the Raman spectrum can serve as a indicator for the estimation of the degree of splitting of SWCNT bundles in the composite.Russian Chemical Bulletin 10/2014; 62(10):2245-2250. DOI:10.1007/s11172-013-0324-6 · 0.48 Impact Factor
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ABSTRACT: The mechanical and thermomechanical properties of metal-containing epoxy composite films based on silver nanoparticles synthesized in situ are investigated. There is a nonmonotonic dependence of the mechanical properties on the concentration of silver myristate used as a precursor. It is found for the first time that the breaking strength and elastic modulus increase by a factor of 1.8–1.5 relative to those of the unmodified matrix at a small concentration of precursor nanoparticles (on the order of 0.1 wt %). DSC and thermomechanical studies reveal that the glass-transition temperature decreases slightly (by 5–6°C) as the precursor concentration is increased to 0.5 wt %, thereby suggesting a weak plasticization of the modified epoxy matrix. On the basis of the spectrophotometry data measured in the region of surface plasmon resonance of silver nanoparticles (420–425 nm) and SEM data, it is inferred that the in situ strengthening of an epoxy nanocomposite based on epoxy resin ED-20, triethylamine, and silver myristate is attained because silver nanoparticles smaller than 20 nm in size and having a narrow particle-size distribution are formed during curing.Polymer Science Series A 05/2014; 56(3):304-310. DOI:10.1134/S0965545X14030031 · 0.92 Impact Factor
Article: Effect of TiO2 nanoparticle additions on the conductivity of network polymer electrolytes for lithium power sources[Show abstract] [Hide abstract]
ABSTRACT: Network copolymer electrolytes were synthesized from polyether (polyester) diacrylates with different structures and chain lengths of polyester diacrylate and polyethylene glycol diacrylate. The optimum matrix for ion transport in the electrolyte was formed from only one type of oligomer. The influence of TiO2 nanopowder additions (∼60 nm) on the conductivity of the copolymer electrolyte was studied. The addition of 10 wt % TiO2 led to an increase in the conductivity by an order of magnitude at 30°C; the effective activation energy decreased by 20%. At elevated temperatures, the mobility of polymer chains increased and the contribution of TiO2 nanoparticles in ion transport was only half of the order of magnitude of the conductivity at 100°C. The increase in the conductivity of the polymer electrolyte after the addition of TiO2 was presumably caused by the formation of a more mobile state of the lithium ion near the nanoparticle surface, as shown by pulsed field gradient (PFG) 7Li NMR.Russian Journal of Electrochemistry 04/2014; 50(4):336-344. DOI:10.1134/S1023193514040107 · 0.76 Impact Factor
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ABSTRACT: The physical and mechanical properties of composite materials based on epoxy-amine systems—diglycidyl ether of diphenylolpropane-eutectic mixture of aromatic amines (40 wt % m-phenylenediamine-60 wt % 4,4′-diaminodiphenylmethane) and epoxy-diane resin ED-20-eutectic mixture of aromatic diamines with ultralow contents (≤0.1 wt %) of single-layer carbon nanotubes—are studied. It is shown that, for the concentration dependence of the modulus of elasticity during tension, the additivity rule is obeyed only at the lowest concentrations of carbon nanotubes. In this case, the presence of near-surface layers of a matrix with an increased elastic modulus should be considered.Polymer Science Series A 06/2011; 53(6):502-509. DOI:10.1134/S0965545X11060071 · 0.92 Impact Factor
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ABSTRACT: With the aim to enhance the crack resistance and impact resilience of heat-resistant epoxy polymeric matrices for high-strength composites, modification of epoxy resins with new poly(ether acrylates) and their adducts with amines was studied.Russian Journal of Applied Chemistry 05/2009; 82(5):894-897. DOI:10.1134/S1070427209050280 · 0.28 Impact Factor
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ABSTRACT: New polymer gel electrolytes based on polyester diacrylates and LiClO4 salt solutions in organic solvents are developed for lithium ion and lithium polymer batteries with a high ionic conductivity up to 2.7 × 10−3 Ohm−1cm−1 at the room temperature. To choose the optimum liquid electrolyte composition, the dependence is studied of physico-chemical parameters of new gel electrolytes on the composition of the mixture of aprotic organic solvents: ethylene carbonate, propylene carbonate, and λ-butyrolacton. The bulk conductivity of gel electrolytes and exchange currents at the gel electrolyte/Li interface are studied using the electrochemical impedance method in symmetrical cells with two Li electrodes. The glass transition temperature and gel homogeneity are determined using the method of differential scanning calorimetry. It is found that the optimum mixture is that of propylene carbonate and λ-butyrolacton, in which a homogeneous polymer gel is formed in a wide temperature range of −150 to +50°C.Russian Journal of Electrochemistry 12/2008; 45(1):101-107. DOI:10.1134/S1023193509010145 · 0.76 Impact Factor
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ABSTRACT: Water-soluble acrylamide monomers N-(hydroxymethyl)acrylamide, N-(hydroxymethyl)methacrylamide, N,N-diethanolacrylamide, N,N-diethanolmethacrylamide, N,N-methylethanolacrylamide, and N,N-methylethanolmethacrylamide have been synthesized and characterized. The kinetics and thermodynamics of the free-radical polymerization of these monomers and of the model compounds N-isopropylacrylamide and acrylamide have been studied by the methods of isothermal and scanning calorimetry. The structure and the solubility of the said polymers in water and organic solvents have been investigated and their molecular-mass characteristics and temperatures of glass transition (T g) and melting (T m) have been examined by DSC, liquid chromatography, 1H NMR and IR spectroscopy, and chemical analysis of functional groups. Hydrogels and amphiphilic network polymers based on acrylamide monomers have been prepared and characterized.Polymer Science Series A 03/2008; 50(3):283-290. DOI:10.1134/S0965545X08030061 · 0.92 Impact Factor
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ABSTRACT: The thermodynamics, kinetics, and mechanism of the anionic polymerization of a number of acrylamide monomers has been studied with the use of isothermal and scanning calorimetry, liquid chromatography, 1H NMR and IR spectroscopy, mass spectrometry, and chemical analysis of functional groups. It has been demonstrated that the polymerization system shows the living character and the interchain exchange reactions are absent. It has been shown that N,N-diethanolacrylamide and N,N-diethanol(meth)acrylamide are uninvolved in anionic polymerization. The causes of this phenomenon have been ascertained. The products of the anionic polymerization of acrylamides are hyperbranched copolymers containing heterochain and carbochain fragments. Macromolecules contain end amide and acrylamide groups; in some macromolecules, end tert-butoxide groups of the used polymerization initiator are detected. For the products of the anionic polymerization of the acrylamide monomers under study, the temperatures of glass transition and melting have been measured.Polymer Science Series A 09/2007; 49(9):987-999. DOI:10.1134/S0965545X07090052 · 0.92 Impact Factor
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ABSTRACT: The mechanism of forming the macromonomer based on ɛ-caprolactone and 2-hydroxyethyl methacrylate under the effect of dibutyltin dilaurate has been investigated. The molecular-mass characteristics and the functional-type distribution of the macromonomer have been determined. It has been established that, as the concentration of the methacrylic component is increased, the contribution of side reactions related to its anionic polymerization rises. It has been shown that macromonomers with caprolactone backbones and terminal methacrylic and hydroxyl groups may be obtained. A method has been proposed for the purification of polymerization products from low-molecular-mass impurities, such as dimethacrylates and diols.Polymer Science Series A 03/2007; 49(3):249-255. DOI:10.1134/S0965545X07030030 · 0.92 Impact Factor
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ABSTRACT: Kinetics and mechanism of equifunctional 4,4′-(N,N′-bismaleimide)-diphenylmethane/2,2′-diallyl-bisphenol A (BMDM/DABPA) and model (phenylmaleimide/2,2′-diallylphenol) (PMI/AP) systems have been studied in the temperature range 140–400 °C using IR-, 1H- and 13C-NMR spectroscopy, gas chromatography–mass spectrometry GCMS), differential scanning calorimetry (DSC) and isothermal calorimetry. It was established that the cure mechanism consists a unique combination of step-wise and chain polymerization and polycondensation reactions: step-wise "ene" addition reaction of allyl group to maleimide one and consecutive/parallel chain polymerization of maleimide and propenyl groups generated by first reaction. The latter reaction is the main crosslinking reaction. The second source of crosslinking is a dehydration reaction of phenol groups that proceeds with mandatory participation as one of the component 1:1 adduct (product of step-wise polymerization). Homopolymerization of maleimide groups proceeds autocatalytically under the action of free radicals generated by thermal decomposition of maleimide-propenyl groups' donor–acceptor pairs. Steric hindrance in 2,2′-diallyl-bisphenol A prevents the reversible Diels–Alder reaction but this reaction proceeds in model systems. Some thermodynamic and kinetic parameters of the reactions are determined. Copyright © 2003 John Wiley & Sons, Ltd.Polymers for Advanced Technologies 10/2002; 13(10‐12):837 - 844. DOI:10.1002/pat.230 · 1.76 Impact Factor
- Mechanics of Composite Materials 08/1991; 26(5):557-561. DOI:10.1007/BF00616630 · 0.47 Impact Factor
- Mechanics of Composite Materials 08/1990; 25(5):582-586. DOI:10.1007/BF00612900 · 0.47 Impact Factor
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ABSTRACT: In our study we establish, using polyepoxides and polyepoxide composites as examples, that the kinetic laws governing the thermal degradation of polymer matrices as a component of composites may differ significantly from those for block polymers, and provide an explanation of this effect.Mechanics of Composite Materials 01/1990; 26(3):278-284. DOI:10.1007/BF00613099 · 0.47 Impact Factor
- Mechanics of Composite Materials 12/1988; 25(1):79-82. DOI:10.1007/BF00608455 · 0.47 Impact Factor
Article: Transformations of active centres during α-oxide polymerizations with complex catalysts containing the FeCl4− anion[Show abstract] [Hide abstract]
ABSTRACT: The polymerization of α-oxides in the presence of R4NFeCl4 and Ar4PFeCl4 was studied. By gamma-resonance spectroscopy it was revealed that an oxidation-reduction reaction proceeds in the course of the polymerization process, resulting in the reduction of trivalent to divalent iron. A reaction mechanism was proposed, and the effect of the α-oxide kind and of temperature on the course of the oxidation-reduction process was investigated.Polymer Science U S S R 12/1988; 30(5):1115-1121. DOI:10.1016/0032-3950(88)90337-1
Article: Polymerization of epoxide compounds under the influence of complex catalysts of the onium type[Show abstract] [Hide abstract]
ABSTRACT: Polymerization of epoxide compounds on exposure to complex catalysts of the onium type has been studied. It is shown that the action of the catalysts is due to their breakdown into active components—Lewis acid and tertiary amine. The MWD and structure of the polymerization products have been studied. It is concluded that the polymerization process studied is cationic in nature.Polymer Science U S S R 12/1987; 29(9):2009-2016. DOI:10.1016/0032-3950(87)90006-2
- Mechanics of Composite Materials 08/1987; 22(5):634-638. DOI:10.1007/BF00714814 · 0.47 Impact Factor
Article: Kinetics of the epoxide binder curing process under conditions of mutual diffusion of the components[Show abstract] [Hide abstract]
ABSTRACT: The kinetics of the curing process in curing epoxide compositions under conditions of mutual diffusion of the reagents is studied on model systems. A relation is obtained for the initial rate of heat evolution as a function of the thickness of the layer of binder components, which is used as the basis for determining the coefficient of mutual diffusion as a function of the degree of conversion. A mathematical model of the process is derived, numerical calculations are made, and the conversion profile for the course of the reaction is obtained.Polymer Science U S S R 12/1986; 28(7):1682-1689. DOI:10.1016/0032-3950(86)90343-6
Article: Production stresses during the solidification of cylindrical articles formed from polymer composite materialsMechanics of Composite Materials 01/1986; 22(1):99-103. DOI:10.1007/BF00606015 · 0.47 Impact Factor
Article: The influence of bridge groups nature in diepoxy compounds on kinetics of curing process by diamines and on physical and mechanical properties of forming polymers[Show abstract] [Hide abstract]
ABSTRACT: In the present work the initial monomer reactivity in curing reaction of epoxy compounds by aromatic diamines has been investigated. Also physical and mechanical properties of cured polumers of diglycidyl ethers 4,4'-bisaromatic compounds with bridge groups have been examined.Polymer Bulletin 04/1981; 4(8):479-485. DOI:10.1007/BF00255704 · 1.44 Impact Factor
Top Journals
Russian Academy of Sciences
Moskva, Moscow, Russia
- • Institute of the Problems of Chemical Physics
- • Institute of Chemistry


text:
|Publication number
|Publication type
|Application number
|Publication date
|Oct 17, 2002
|Filing date
|Jan 9, 2001
|Priority date
|Jan 9, 2001
|Also published as
|Publication number
|Tobin Hellyer, Qimin You, James Harris
|Original Assignee
|Hellyer Tobin J., Qimin You, Harris James M.
|Export Citation
|BiBTeX, EndNote, RefMan
|Classifications (8), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
[0001] The present invention relates to materials and methods for detection of HIV nucleic acids, in particular to probes and primers for detection of HIV in hybridization and amplification assays.
[0002] The genome of the Human Immunodeficiency Virus (HIV) is highly heterogeneous and exhibits a mutation rate on the order of 10−4 per base per generation. Combined with the rapid rate of viral propagation in infected individuals, this presents particular challenges for diagnostic nucleic acid amplification techniques which typically amplify a single conserved sequence within a target organism. Although a great deal of research activity has been directed to detecting HIV-1 in hybridization and amplification assays, such molecular assays have so far been limited by their inability to detect all genotypes with equal efficiency. Although a signal amplification system for the detection of HIV-1 type O has been reported, none of the currently available diagnostic nucleic acid amplification methods are able to detect this genotype of the virus.
[0003] The present invention provides primers and probes derived from the HIV-1 pol gene which facilitate detection and/or quantification of all presently known genotypes of HIV-1 (A-I and O). A single amplification primer pair according to the invention efficiently amplifies all known genotypes of HIV-1, which may then be detected in a single detection step using the detector probes and primers of the invention.
[0004]FIG. 1 shows the titration of internal control fluorescent signal (FAM) with increasing levels of native HIV target (ROX).
[0005] The primers, hybridization probes and detector probes of the present invention are complementary to regions of the HIV-1 polymerase (pol) gene. Initially, design of the disclosed primers and probes was based on conserved regions in an alignment of fifty-eight HIV-1 pol sequences. Additional data was subsequently added to the alignment to provide a database with a total of 115 pol sequences. One goal was to develop probes and primers which, in spite of the heterogeneity of the pol sequence, provided amplification, detection and/or quantitation of all presently known HIV-1 genotypes with essentially equal efficiency in amplification reactions. In some cases this was accomplished by overlapping the hybridization site of the 5′ ends of certain of the detector probes with the hybridization site of the 3′ end an amplification primer. This approach took advantage of sequence conservation in the SDA primer region and avoided much of the sequence heterogeneity evident in the intervening region between the two SDA primers. This technique also allowed use of a smaller target sequence, thereby improving amplification efficiency.
[0006] As used herein, an amplification primer is an oligonucleotide for amplification of a target sequence by extension of the oligonucleotide after hybridization to the target sequence or by ligation of multiple oligonucleotides which are adjacent when hybridized to the target sequence. At least a portion of the amplification primer hybridizes to the target. This portion is referred to as the target binding sequence and it determines the target-specificity of the primer. In addition to the target binding sequence, certain amplification methods require specialized non-target binding sequences in the amplification primer. These specialized sequences are necessary for the amplification reaction to proceed and typically serve to append the specialized sequence to the target. For example, the amplification primers used in SDA include a restriction endonuclease recognition site 5′ to the target binding sequence (U.S. Pat. Nos. 5,270,184 and 5,455,166). NASBA, 3SR and transcription based amplification primers require an RNA polymerase promoter linked to the target binding sequence of the primer. Linking such specialized sequences to a target binding sequence for use in a selected amplification reaction is routine in the art. In contrast, amplification methods such as PCR, which do not require specialized sequences at the ends of the target, generally employ amplification primers consisting of only target binding sequence.
[0007] As used herein, the terms "primer" and "probe" refer to the function of the oligonucleotide. A primer is typically extended by polymerase or ligation following hybridization to the target but a probe typically is not. A hybridized oligonucleotide may function as a probe if it is used to capture or detect a target sequence, and the same oligonucleotide may function as a primer when it is employed as a target binding sequence in an amplification primer. It will therefore be appreciated that any of the target binding sequences disclosed herein for amplification, detection or quantitation of HIV-1 may be used either as hybridization probes or as target binding sequences in primers for detection or amplification, optionally linked to a specialized sequence required by the selected amplification reaction or to facilitate detection.
[0008] Based on the alignment of multiple HIV-1 pol gene sequences, the following amplification primers were designed for testing in SDA reactions. Target binding sequences are underlined. The remaining 5′ portion of the sequence comprises the restriction endonuclease recognition site (RERS) that is required for the SDA reaction to proceed and a generic non-target-specific tail sequence. It will be readily apparent that the target binding sequences may be used alone to amplify the target in reactions which do not require specialized sequences or structures (e.g., PCR) and that different specialized sequences required by amplification reactions other than SDA may be substituted for the RERS-containing sequence shown below (e.g., an RNA polymerase promoter). "R" and "L" in the primer name indicates "right" and "left" primers, respectively, when the oligonucleotides are used in amplification reactions:
[0009] In addition, the following detector primers were designed for real-time detection of amplification products produced using the amplification primers. The structure and use of such detector primers is described, for example, in U.S. Pat. No. 5,547,861 and U.S. Pat. No. 5,928,869. The target binding sequences are underlined. The remaining portion of the sequence forms a hairpin structure which is typically labeled to facilitate detection of amplification products as is known in the art. It will be readily apparent that the target sequence may be used alone for direct detection by hybridization (typically linked to a detectable label) and that other directly and indirectly detectable sequences and labels may be substituted for the hairpin as is known in the art. See, for example U.S. Pat. No. 5,935,791; U.S. Pat. No. 5,846,726; U.S. Pat. No. 5,691,145; U.S. Pat. No. 5,550,025 and U.S. Pat. No. 5,593,867.
DETECTOR PRIMERS QDR66 TAGCACCCGAGTGCTGGCAAATTCATTTCTTCTAATACTG SEQ ID NO:11 QDR64 TAGCACCCGAGTGCTGCAAATTCATTTCTTCTAATACTGT SEQ ID NO:12 QDR56 TAGCACCCGAGTGCTAAATTCATTTCTTCTAATACTGT SEQ ID NO:13 QOL1 TAGCACCCGAGTGCTAGGAGCAGATGATACAGT SEQ ID NO:14 QOL2 TAGCACCCGAGTGCTCAGGAGCAGATGATACAGT SEQ ID NO:15 QOL3 TAGCACCCGAGTGCTACAGGAGCAGATGATACAGT SEQ ID NO:16 QOL4 TAGCACCCGAGTGCTGAGCAGATGATACAGT SEQ ID NO:17 QOL5 TAGCACCCGAGTGCTGGAGCAGATGATACAGT SEQ ID NO:18 DL56HPD TAGCACCCGAGTGCTCACAATGTTAAAAGAAAAGGG SEQ ID NO:19 DL52HPD TAGCACCCGAGTGCTACAATGTTAAAAGAAAAGGG SEQ ID NO:20 DL50HPD TAGCACCCGAGTGCTCAATGTTAAAAGAAAAGGG SEQ ID NO:21 DR58HPD TAGCACCCGAGTGCTCCCCTTTTCTATTAAAATTGTG SEQ ID NO:22 DR54HPD TAGCACCCGAGTGCTCCCTTTTCTATTAAAATTGTG SEQ ID NO:23 DR52HPD TAGCACCCGAGTGCTCCCCTTTTCTATTAAAATTG SEQ ID NO:24 DR48HPD TAGCACCCGAGTGCTCCCTTTTCTATTAAAATTG SEQ ID NO:25 Pol2DR58 TAGCACCCGAGTGCTCCCCTTTTCTTTTAAAATTGTG SEQ ID NO:26 DN2 TAGCACCCGAGTGCTCCCAATCCCCCCTTTTCTGTTAAAAT SEQ ID NO:27 DN3 TAGCACCCGAGTGCTCCCCAATCCCCCCTTTTCTGTTAAAAT SEQ ID NO:28 DN4 TAGCACCCGAGTGCTCCAATCCCCCCTTTTCTGTTAAAAT SEQ ID NO:29 DN5 TAGCACCCGAGTGCTCAATCCCCCCTTTTCTGTTAAAAT SEQ ID NO:30 DN5.1 TAGCACCCGAGTGCTCAATCCCCCCTTTTCTTTTAAAAT SEQ ID NO:31 OL62 TAGCACCCGAGTGCTCCCAATCCCCCCTTTTCTTTT SEQ ID NO:32 OL64 TAGCACCCGAGTGCTCCCAATCCCCCCTTTTCTTTTA SEQ ID NO:33
[0010] SEQ ID NOs:11-13 and 19-26 are conventional non-overlapping detector primers which contain a hairpin as described in U.S. Pat. No. 5,928,869. SEQ ID NOs:14-18 and 27-33 also contain the hairpin but the 5′ end of the target binding sequences overlap with the 3′ end of the target binding sequences of the upstream amplification primers.
[0011] Bumper primers used in SDA (BR and BL) were also designed. The entire sequence of these oligonucleotides consists of target binding sequence:
[0012] The sequences set forth above were selected to minimize the effects of heterogeneity in the targeted region of the pol gene. Mismatches were confined to the middle or the 5′ end of the sequences to permit efficient 3′ extension upon hybridization to the target sequence. Only three of the 117 HIV-1 strains analyzed exhibit more than one mismatch with amplification primers SEQ ID NO:5 and SEQ ID NO:9 or with detector SEQ ID NO:26. Detector SEQ ID NO:27-30 contain a deliberate mismatch eight bases from the 3′ end to minimize target-specific variations in priming efficiency caused by heterogeneity in the target region.
[0013] Because the target binding sequence confers target specificity on the primer or probe, it should be understood that the target binding sequences exemplified above for use as particular components of a specific amplification reaction may also be used in a variety of other ways for detection of HIV. For example, the target binding sequences of SEQ ID NOs:1-37 may alternatively be used as hybridization probes for direct detection of HIV-1, either without prior amplification or as a post-amplification assay. Such hybridization methods are well known in the art and typically employ a detectable label associated with or linked to the target binding sequence to facilitate detection of hybridization. Further, essentially all of the target binding sequences set forth above may be used as amplification primers in amplification reactions which do not require additional specialized sequences (such as PCR) or appended to the appropriate specialized sequences for use in 3SR, NASBA, transcription-based or any other primer extension amplification reactions. For detection of amplification products, amplification primers comprising the target binding sequences disclosed herein may be labeled as is known in the art, or labeled detector primers comprising the disclosed target binding sequences may be used in conjunction with the amplification primers as described in U.S. Pat. No. 5,547,861 and U.S. Pat. No. 5,928,869 for real-time homogeneous detection of amplification. Such detector primers typically comprise a directly or indirectly detectable sequence which does not initially hybridize to the target but which facilitates detection of the detector primer once it has hybridized to the target and been extended. For example, such detectable sequences may be sequences which form a secondary structure, sequences which contain a restriction site, or linear sequences which are detected by hybridization of their complements to a labeled oligonucleotide (sometimes referred to as a reporter probe) as is known in the art. Alternatively, the amplification products may be detected post-amplification by hybridization of a probe selected from any of the target binding sequences disclosed herein which fall between a selected set of amplification primers.
[0014] It is to be understood that an oligonucleotide according to the invention which consists of a target binding sequence and, optionally, either a sequence required for a selected amplification reaction or a sequence required for a selected detection reaction may also include certain other sequences which serve as spacers, linkers, sequences for labeling or binding of an enzyme, etc. Such additional sequences are typically known to be necessary to obtain optimum function of the oligonucleotide in the selected reaction and are intended to be included by the term "consisting of."
[0015] SDA reactions were performed to determine the analytical sensitivity of the assay for the detection of HIV DNA. Amplification was carried in the presence of 0, 5, 10, 50, 100 or 250 copies of HIV target DNA. The target sequence was a fragment of the HIV-1 genome corresponding to nucleotides 4659-4910 of strain B-WEAU (GenBank accession number U21135) that was cloned into the plasmid vector pBlueScript SK+ (Stratagene). SDA was performed at 52� C. using 500 nM primers (SEQ ID NO:5 and SEQ ID NO:9), 50 nM bumpers (SEQ ID NO:36 and SEQ ID NO:37), and 200 nM detector probe, SEQ ID NO:26, in buffer containing: 100 mM bicine; 30 mM potassium hydroxide; 68 mM KiPO4, pH7.6; 10.5% glycerol; 6.5% DMSO; 0.7 mM dCTP; 0.1 mM dA-, dG- and dTTP; 70 ng/μl human placental DNA; 100 ng/μl bovine serum albumin; 4 mM magnesium acetate; 6U Bst polymerase and 32U BsoBI restriction enzyme. The detector primer was labeled at the 5′ terminus with a fluorescein donor molecule and internally with a dabcyl quencher moiety. The two dyes on the detector were separated by BsoBI restriction endonuclease recognition sequence and held in close juxtaposition by a hairpin structure within the tail sequence as described in U.S. Pat. Nos. 5,919,630; 5,928,869 and 5,958,700. Donor fluorescence was monitored during the course of amplification.
[0016] In the presence of target, donor fluorescence increased during the course of the reaction as the hairpin holding the donor and quencher in close proximity unfolded and the restriction site was cleaved. In contrast, in the absence of target, fluorescence remained consistently low throughout the reaction. Results were expressed in terms of area under the curve or "MOTA." A larger MOTA score indicates generation of more fluorescence and, generally, the presence of more input target. The results are shown in Table 1.
TABLE 1 TARGETS PER MEAN MOTA REACTION SCORE (n = 28)* 0 606 5 14883 10 20565 50 47643 100 56740 150 61676
[0017] The limit of detection (LOD), defined as the input target level at which 95% of reactions would yield a positive result, was determined to be between 9 and 11 copies of target DNA depending on the MOTA score selected as a cut-off for determining a positive result. These results demonstrate that the amplification and detector primers are capable of sensitive and reproducible detection of HIV DNA.
[0018] A similar experiment to that described in Example 1 was performed using the SEQ ID NO:27 detector primer, the target hybridization region of which overlaps the SEQ ID NO:9 amplification primer. This probe was designed to take advantage of sequence conservation in the amplification primer binding region while maintaining the specificity afforded by detection of the internal region of the SDA amplicon that lies between the two amplification primers. Buffer conditions were the same as described above with the following modifications: 75 mM KPO4, 14% DMSO, 5% glycerol and 27U BsoBI. The limit of detection using the overlapping probe was 61 to 86 copies of HIV target DNA depending on the MOTA score selected for determining a positive result. These results demonstrate sensitive and specific detection of HIV target DNA using an alternative probe design that offers additional flexibility in the development of SDA-based systems.
[0019] The SDA assay for HIV DNA was converted to a two-step reverse transcriptase (RT)-SDA format in which RNA was first copied to cDNA using an RT enzyme and then amplified in a conventional SDA reaction. One important difference between the SDA conditions described in Examples 1 and 2 and the RT-SDA described here is the absence of bumper primers. The need for bumpers apparently was precluded by the choice of an RT enzyme which possesses RNase H activity and which is therefore capable of degrading the RNA template following reverse transcription, thereby liberating a single stranded DNA target sequence in to solution. An LOD study was performed for HIV RNA using in vitro transcripts of a genotype B strain generated from the pBlueScript plasmid clone described in Example 1.
[0020] Reverse transcription was carried out in microtiter wells using 15U avian myeloblastosis virus (AMV)-RT in buffer containing: 69.3 mM bicine; 12.3 mM KOH; 20.8 mM KiPO4; 3% glycerol; 4.5% DMSO; 6 mM magnesium acetate; 2.75 mM dCsTP; 0.25 mM dA-, dG- and DTTP; 100 ng/μl BSA; 1250 nM SEQ ID NO:5 and 700 nM SEQ ID NO:9. In brief, the reverse transcription reaction mixture without enzyme was incubated for 10 min at 68� C. to denature the target RNA. Eighty microliters of denatured target was then added to a microwell containing 20 μl of an RT enzyme mixture that was pre-equilibrated at 50� C. The complete RT reaction was incubated for 10 min to facilitate synthesis of cDNA and degradation of the RNA template by the RNase H activity of the RT enzyme. To initiate amplification, 40 μl of the reverse transcription reaction was transferred to a second microtiter well at 52� C. containing 60 μl of an amplification mixture comprising Bstpolymerase, BsoBI restriction enzyme, and fluorescent detector primer together with certain buffer components. The final SDA conditions were as follows: 72 mM bicine; 24 mM potassium hydroxide; 54.4 mM KiPO4; 4% glycerol; 11% DMSO; 0.1 mM dA-, dG- and dTTP; 1.1 mM dCTP; 700 ng/μl hpDNA; loong/μl BSA; 500 nM SEQ ID NO:5; 300 nM SEQ ID NO:9; 600 nM SEQ ID NO:26; 12U BsoBI and 7U Bst polymerase. The wells were sealed and incubated at 52� C. Donor fluorescence was monitored throughout the course of the reaction.
[0021] The results of this experiment are shown in Table 2.
TABLE 2 RNA TARGETS MEAN MOTA PER REACTION SCORE (n = 17) % POSITIVE 0 139 0 10 3577 64.7 25 7810 88.0 50 13410 100.0 75 23641 100.0 100 35719 100.0 500 73870 100.0
[0022] MOTA scores ≧1000 were considered positive . All reactions containing ≧50 copies of HIV target RNA were positive as were 88% containing 25 copies and 65% of those containing 10 copies. These results demonstrate the sensitivity of the disclosed amplification and detector primers for the detection of HIV-1 RNA.
[0023] RT-SDA was performed on purified RNA from representative isolates of nine different clades of HIV-1 (Boston Biomedica, Inc.). In order to quantify the parental stocks of viral RNA, all except the Type O nucleic acid were tested with each of two commercially available quantitative HIV tests: the Roche Amplicor HIV-1 Monitor V1.5 and the Chiron Quantiplex HIV-1 RNA 3.0 Assay. Since neither of these tests is able to amplify RNA of HIV-1 genotype O, a third non-amplified system, the Digene Hybrid Capture Assay, was used to quantify the type O nucleic acid. Viral RNAs for genotypes A-H were diluted according to the results of the Roche assay to give the equivalent of 200 copies per RT-SDA reaction. Type O RNA was diluted to the same level based on the results of the Digene test. RT-SDA was performed according to the method described in Example 3. For eight of the nine clades, all sixteen replicates were positive at 200 copies per RT-SDA reaction. For genotype F, {fraction (4/16)} (25%) replicates were positive. The reason for the apparently lower sensitivity with type F is unclear but might be attributed to inaccurate qualification of the parental RNA stock of this genotype by the Roche assay. Alternatively, the discrepancy might be due to the presence in the type F sequence of a single base mismatch with the right amplification primer which is used to initiate first strand cDNA synthesis (SEQ ID NO:9). Importantly, all reactions conducted with 2000 input copies of type F RNA were psotive.
[0024] These data demonstrate that the disclosed primers and probes are capable of detecting multiple genotypes of HIV-1, including type O, with a high degree of sensitivity. Detection of type O RNA is particularly important in view of the inability to detect this lade with the majority of commercially available diagnostic nucleic acid assays.
[0025] In vitro transcripts generated from the plasmid clone described in Example 1 were quantified by competitive RT-SDA. In brief, RT-SDA was performed as described above with the exception that two detector primers were included in the reaction mixture, both at a concentration of 200 nM. The first primer, SEQ ID NO:26, was specific for HIV-1 and was labeled at the 5′ end with dabcyl quencher moiety and internally with rhodamine (Rox). The second probe was specific for an internal control sequence and was labeled at its 5′ end with fluorescein and internally with a dabcyl quencher. The internal control comprised an RNA molecule that was generated by in vitro transcription of a mutated clone of the HIV-1 pol gene. The internal control possessed the same primer binding regions as the native HIV target but differed by a series of point mutations introduced to coincide with the 3′ end of the detector hybridization region. These mutations permitted discrimination of the native target and internal control by preventing hybridization and extension of the mismatched probes during amplification. The internal control was designed such that it amplified with similar efficiency to the native target and would compete during the course of the reaction for one or more rate-limiting reagents. The same amount of internal control was seeded into each test sample and into a series of calibrator reactions containing known amounts of native target. Details of the theoretical aspects of the quantitative SDA algorithm may be found elsewhere (J. G. Nadeau, et al. 1999. Anal Biochem 276, 177-187; C. M. Nycz, et al. 1998. Anal Biochem 259, 226-234). In brief, the fluorescence produced by amplification of both the native target and internal control was monitored at discreet intervals throughout the reaction. Data were processed through a series of normalization and smoothing functions to produce a fluorescent signal for both sequences. From these values, the natural log of the ratio of native target and internal control signals was calculated. The values obtained from the calibrator wells were plotted as a regression against the natural log of the input number of target molecules of RNA to produce calibration curves corresponding to each time point. An automated algorithm was then used to determine the time at which it was statistically optimal to perform quantification.
[0026]FIG. 1 shows the MOTA scores for the internal control with various copy numbers of input target sequence. MOTA scores for the internal control decreased with increasing levels of native target, demonstrating the competition between the two targets that is necessary for accurate quantification. Table 3 shows the results of quantitative competitive RT-SDA with between 50 and 100000 copies of native RNA target and 5000 copies of internal control per reaction. At all but one target level, accuracy and precision were better than �25%, thus demonstrating the ability to quantify HIV-1 RNA using the disclosed amplification and detector primer sequences.
TABLE 3 TARGET RNA COPIES/REACTION REVERSE PERCENT TRANSCR. SDA* MEAN (n = 8) ACCURACY PRECISION 0 0 16 62.78 125 50 47 −5.17 25.47 1250 500 537 7.45 24.19 12500 5000 8765 75.29 9.07 125000 50000 60009 20.02 5.48 250000 100000 76351 −23.65 7.26
[0027] Sequence alignment data and initial testing of the additional target binding sequences disclosed herein indicates that similar results would be obtained using these sequences in probes and primers for amplification and/or detection of all genotypes of HIV-1.
|U.S. Classification
|435/5, 536/23.72, 435/6.14
|International Classification
|Cooperative Classification
|European Classification
|Jan 9, 2001
Owner name: BECTON, DICKINSON AND COMPANY, NEW JERSEY
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Paula Deen Stainless Steel Signature Collection 12-Inch Nonstick Skillet
Ya'll can depend on the Paula Deen Stainless Steel Signature Collection for even heating and browning but you may have to stir your collard greens as they simmer. While this deep straight-sided skillet is good for pan-fries, it's not an ideal choice for omelets. You will have to exert a little elbow grease to remove stains too and you'll need to pull out the copper cleaner to clean the copper inserts in the bottom. A nice bonus: It comes with a Paula Deen recipe.
Type: Nonstick
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Price When Reviewed: $49.99
- Distributes heat very well on gas and electric ranges
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October 7, 2015
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How to fix common mistakes in early intervention documentation.
AOTA asked Medicare to rescind a restrictive proposal on lower-limb prosthetics.
Register today for the AOTA/NBCOT National Student Conclave.
Specialty Certification in Environmental Modification is for occupational therapists and occupational therapy assistants who modify, adapt, or change contexts – especially the environments of home, school, work, or community. Practitioners determine environmental needs and provide intervention strategies, devices, space adaptations and/or assistive technology that reflect the person-environment fit and address occupational performance that maximizes participation, safety, accessibility, and independence.
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San Pedro, CA
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Upsidedown Brown
Joe Schofield
August 3rd, 2012
Viewed 343 Times - Last Visitor from Wilmington, DE on 10/12/2015 at 10:16 PM
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More from Joe Schofield
Yellowknife, NT - Canada
Great shot Joe love the detail you got
Much appreciated, Darren !
Fullerton California, ca - United States
Great image...v/t/p
Thank you, Athala !
San Miguel, CA - United States
A beautiful fallen leaf shot. Voted.
Thank you, Susan !
Northridge, CA - United States
Love the color and the shadows effect on this amazing shot..V,F,T)
Thank you Bedros!
Spokane, WA - United States
Great image Joe, I love the textures in the leaf as well as the groung, the shadows from the water droplets are fantastic, great job!v
Thx, Kari!
Franklin Square, NY - United States
Joe, great idea for a photo shoot, love the dried up leave laying upside down, with the morning dew on it. Very detail, and the clarity is fantastic. Nice background piece to go with it. Nice shot, voted, Tweet, FB and Google + promoted
Thank you, John !
Dubai - United Arab Emirates
Picture Perfect!!! Love the title which reflects it too. Congrats!
Bury, Ma - United Kingdom
Fantastic image Joe. I love the how the lighting has revealed the texture and how the leaf's shadow looks like an old boot. Voted
Plainview, TX - United States
nice catch!
Beverly Hills, CA - United States
very nice and unusual take v
Brussels - Belgium
Great fall image. I like the details of the waterdrops. V!
Catskill, NY - United States
Wonderful capture of the water drops on a dead leaf. Great textural detail and shadow. v/fb
Thanks, Ellen!
Rochester, NY - United States
Nice shadows.
Tucson, AZ - United States
Beautiful...really like this.
Redan, vi - Australia
wonderful capture V
Scotts Valley, CA - United States
Great details, capture, and monochromatic! Voted, pin it, g+1
Woodburn, OR - United States
I am a water droplet nut! I love water drops and you captured them with so much beauty. The shadows are perfect, the framing is perfect, the monochromatic color of everything is perfect - I love it.
: ) water drop nuts of the world unite ! Thank you Helen !
Suffolk, VA - United States
Great capture, love all the different textures. v
Prescott, AZ - United States
Very nice...voted.
Hecla, SD - United States
A great capture of this leaf, with wonderful brown tones, textures and details! F/V
Montreal, QC - Canada
lovely aging leaf!
Perham, MN - United States
Leaves have always fascinated me and this capture is just wonderful! Beautiful shot! v
Calgary, AB - Canada
Beautiful - voted!
Calgary, AB - Canada
I love this Joe! The water drops, the light and shadows, the texture of the background... all beautifully captured! voted :)
Holbrook, MA - United States
Brillian capture Joe - love the colors, composition and the details of this image -well done! voted, faved, liked on Facebook & g+ promoted
Thank you so much, Alex !
New Orleans, La - United States
I like the simplicity of the piece, great texture and beautiful browns voted and tweet
JABALPUR, MA - India
The image with presentation of beauty of nature.....Joe..f/v (i feel to grab for my creativity ,,,??)
Jacksonville, FL - United States
nicely done !! v
Auburn, PA - United States
Super detail in this photograph Joe - Great lighting - Dave - V/F/P
Rochester, NY - United States
very cool.....upside down, shading at the top instead of bottom, water on a dried leaf....like the richness of these colors...v/f
Morro Bay, CA - United States
Very interesting and unique!!
Thunder Bay, ON - Canada
Joe I like everything about this photo, the earth tones, the water droplets, the shadow... this would look great on a wall. Voted and Favorite
Much appreciated Sue !
Astoria, NY - United States
Great textures, details, sharpness and lighting
Calgary, AB - Canada
Beautiful detailed capture! vf
Maryland Heights, MO - United States
The detail is amazing! Voted.
Chicago, IL - United States
Perfect title! what a great composition...same tones, different textures. Also love the drops of water! v/t
Concho - Snowflake, AZ - United States
Voted Texturally brilliant, and really like the richness of the colors
Young Harris, GA - United States
Love the lighting....the water drops and shadows are great! V
Galveston, TX - United States
Great macro very fall like, love the water droplets! :D V
Port Vincent, LA - United States
Amazing capture!!!! I really love all the different grains in that capture. And it turned to be almost monochrome.... That's ART!!! LOVE it, - Faved - Voted - Pinned - and Tweet :)
Very much appreciated Anne !
Rochester, Ne - United States
Beautiful capture Joe!!!
Seattle, WA - United States
Very creative capture! v. Marcie
Mandeville, LA - United States
Wonderful image!
Clovis, NM - United States
Interesting title and treatment; turning the image upside down.
Toronto, ON - Canada
Joe this is wonderful image in harmony and nice in detail v/f
Lexington Park, MD - United States
The water droplets on the leaf is a great detail! Good one Joe!
Thanks Megan!
Laguna Hills, CA - United States
Joe, it's amazing how something as simple as a leaf can look so lovely! R21vp
Thank you Enzie!
Long Prairie, MN - United States
Great photograph Joe. Love how can see the veins in the leaf, and like your lighting creating interesting shadows of the leaf and the droplets. v/f
Thank you Bonnie !
Naples, FL - United States
Very nice...
Murphy, NC - United States
Very nice. awesome details
Please Wait...
Beverly Hills, CA (United States)
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Description: The WAV Trigger is a unique high-fidelity polyphonic audio player with surprising capabilities. Supporting up to 2048 uncompressed 16-bit, 44.1kHz wav files – the same quality as an audio CD – the WAV Trigger can play and mix up to 14 stereo tracks simultaneously and independently, with very low latency. Tracks can be controlled via 16 programmable trigger inputs, or by using a native serial control protocol or even MIDI.
Trigger inputs can be connected directly to switches and buttons, or to digital outputs from sensors or another microcontroller. Alternate functions can be specified using a free cross-platform GUI application, and allow triggers to play sequential or random tracks, pause and resume groups of tracks and even control volume. An Arduino library allows for complex serial control like real-time mixing, starting multiple tracks in sample-sync and smooth cross-fading between tracks.
On-board sample rate conversion allows for smoothly changing playback speed/pitch from 0.5x to 2x. in real-time.
MIDI allows you to use the WAV Trigger as a polyphonic sampling synthesizer to play your own sounds from any MIDI keyboard controller. MIDI Channels and Note numbers are mapped to track numbers, and MIDI Controllers adjust volume as well as attack and release times. MIDI Program Change is supported to switch between up to 16 banks of 128 sounds. The WAV Trigger audio engine even implements, pitch bending, voice stealing (oldest playing voices are used for new MIDI Notes when all 14 voices are being used), note attack (fade-in), note release (fade-out) and latency averages 8 ms.
The WAV Trigger supports both SDSC (up to 2GB) and SDHC (up to 32GB) type microSD cards.
Check the link in the documents below to keep up with the latest Firmware updates!
Note: This product is a collaboration with Robertsonics. A portion of each sales goes back to them for product support and continued development.
Anticipating the first question… This version just updates the shipping firmware to the latest rev (v1.21) and updates the above product description with the latest firmware feature set. New capabilities include support for 2048 tracks, real-time playback speed/pitch control and enhanced trigger and MIDI features. It's no different than if you had bought the previous version and updated the firmware.
Also, here's the link to the Arduino serial control library, and here's a short tutorial on serial control with an Uno.
GREAT little board.. We have used over 30 of these since January 2015.. Very powerful… Lots of applications.. GREAT JOB ROBERTSONICS !!
I would like to hook up a midi keyboard, but it has more than 16 keys, so how could I play more than sixteen notes of a single instrument? Does this support looping of sounds as in the case of holding down a key? Also, what's a good source to download instrument samples? Thanks
The WAV Trigger supports up to 2048 tracks. When the serial port is put into MIDI mode, midi channels and note numbers are mapped to track numbers as follows (with the current firmware): Track = (MIDI Channel * 128) + MIDI Note Number.
Because SD card memory is not an issue, I advise you to loop your sounds in the WAV file and just make the tracks longer than you'd ever hold a note. The WAV Trigger does not currently support arbitrary loop points or seamless looping - looping is always over the entire track and there can be a small (< 5ms) gap at the loop point.
Here's a tutorial with links to some Mellotron sound files. Any instrument sample sets that you can load into a sound editor are potentially good to use with the WAV Trigger. I make my own using soft synths.
Is there any way to send the audio output through wires instead of using the 3.5mm jack? I'm sending signal to wire inputs on a class-D amplifier, and I don't have enough room to fit a 3.5mm plug. I searched the hookup guide, user guide, and datasheet and couldn't find an answer.
Not using an existing connector. You could always just solder 3 wires to the pins/pads of the 3.5mm jack.
Haha, I was afraid that would probably be the only option. I'll give it a try, thanks!
I was wondering if it would be possible to take advantage of the Bank Up/Down when using regular triggers.
I've re-purposed an old MIDI pedalboard to launch samples (which works great so far), but since the number of pedals is limited, using Bank Up/Down could allow me to expand the number of samples.
Any chance that could be implemented in a future update (if not already doable)?
Hey there! Looks like a fun little board. One question, though (and please keep in mind I'm new at this). Is it possible to trigger these sounds via remote control? I'm currently building an R2D2 robot and looking for a way to play sounds. This board looks good but I'd need to be able to trigger the sounds from a remote control and also in response to onboard signals from the R2 (i.e. if the proximity detector sees an obstruction, tell the WAV trigger to play a sound).
Any suggestions on a (preferably easy) way to accomplish this?
Hi, that would be very achievable with this board. Your best option would be to have two microcontrollers (like Arduino) talking to each other over the wireless solution of your choice (I prefer Bluetooth, but you could use XBees, RF, WiFi, etc). On one end you could have your buttons and switches. On the other end, the micro is connected to the wireless transceiver as well as the WAV trigger. A button press on one end could be transmitted over the air, to the other micro, which would then pull the corresponding pin low on the WAV trigger. It's also possible to fine a wireless solution that has it's own GPIO built it, so you could circumvent the microcontroller altogether.
Thanks! Upon further looking I may decide to go with the MP3 board just for more storage/triggers (R2 has a lot of sounds!). But your explanation still holds. Incoming order soon!
You mean like this?
The WAV Trigger holds 2048 tracks vs the MP3 Trigger's 255. There are few things the MP3 Trigger can do that the WAV Trigger can't do better. Just saying.
Actually, you could connect an XBee S1 module directly to the WAV Trigger's RX pin and send serial commands over XBee with no on-board Arduino. Just a thought.
Ha! You know…. something like that. :)
Thanks for the link!
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I've been looking for a music synthesizer for several of my projects and found the existing MIDI synth chips less than pleasant to listen to; lots of noise and poor sound quality. The WAV Trigger is simply amazing for me. To be able to select among 2k sounds, at CD quality, and play up to 12 at once for less than $50 is simply amazing. The software to test and change the trigger options is great. You can even operate the board without a microprocessor by connecting to the 16 channels of trigger input. The only additional feature I'd ask for is to be able to dynamically change the balance (the relative Right/Left amplitude). I think this board will keep me busy for the next 6 months of research and development.


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The Swedish Nitro Team had another happy snowboarding year. This is the video that came out from winter 2009/10 riding. PART 1
Riders PART 1: Anton Gunnarsson, Mattias Nyberg, Johannes Skottheim.
Riders PART 2:Anton Bilare, Nils Arvidsson, Tobias Karlsson, Johannes Skottheim, Sven Thorgren, Martina Larsson, Mikael Sandy and Jonas Hagström
Edit: Magnus Törnkvist
Intro: Chris Thelin