text:
Men's Dr. Martens 6" Ironbridge Industrial Grizzly Boots, Black
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Dr. MartensTM 6" Ironbridge Industrial Grizzly Work Boots For Men "Bear" - ish work boots on tough work conditions!
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text:
'I was attracted to him, so I enjoyed it': Teen who auctioned off her virginity for £8,800 reveals details of her first time
Student Alina Percea auctioned off her virginity to the highest bidder on a German website
A teenager who sold her virginity online for £8,800 has revealed the details of her tryst with the winning bidder.
Alina Percea, 18, auctioned her virginity on a website so that she could afford to pay for her computing degree.
The winner of the auction was a 45-year-old Italian businessman but she had no qualms about going through with the deal.
The businessman from Bologna paid for her to fly to Venice where the couple toured the sights before spending a night in a luxury hotel.
Alina, who underwent two medical examinations to prove her virginity, said: 'I liked the man and got on with him well. He didn't look 45, and he seemed much younger.
'We spoke in English as I can't speak Italian and he can't speak Romanian. He paid me a lot of compliments throughout the day, and he was very funny and charming. We got on very well, and I was pleased he'd won.
He told me he had a good job, but he didn't say if he was married or had a family, and I didn't ask him.'
Alina left her home town of Caracal, Romania, in January with two cousins, to find work in Mannheim, Germany, but lost her job in a restaurant because her German was too poor.
She was inspired to auction herself on a German erotic website after reading how American woman Natalie Dylan, 22, from San Diego, had put her virginity up for sale for £2.5million.
The Romanian had hoped to raise as much as £50,000 through the controversial sale but still plans to go to university.
'The bidding lasted two weeks,' she told Closer magazine.
'It started off at 90p but within a couple of days it was almost at £4,400.'
'The winner was a new bidder, who appeared right at the end of the auction.
'He said he would fly me to Venice to meet him.'
The auction ended at £8,800 (10,000 euros).
Alina Percea was inspired to auction herself on a German erotic website after reading how American woman Natalie Dylan (pictured) had put her virginity up for sale for £2.5million
'When the auction ended, I emailed the winner, a 45-year-old Italian business man, to congratulate him.'
'I hoped I'd meet a nice man, like in the film Pretty Woman,' Alina says.
Alina Percea had unprotected sex with her businessman lover
'At the arrivals lounge, a man came over, smiled, handed me a box of chocolates and said: "Welcome to Venice." He looked much younger than 45, short, but nicely dressed, with dark hair, green eyes and a kind smile.'
The two went sightseeing, then to a five-star hotel where they had unprotected sex.
'We kissed, then undressed each other,' Alina says. 'I'd never done that before, so I was nervous. He laid me on the bed and started kissing my body, then we had sex. I was attracted to him, so I enjoyed it, even though it was quite painful.
'We only had sex once, then fell asleep. Next morning, we had breakfast together like any other couple, and I took the morning-after pill. He told me he'd like to see me again, and I agreed.'
But the money raised from the auction was far less than the amount Alina had hoped to make.
'Ten thousand euros is not so much money, even in Romania,' admitted Alina. 'But it will be enough.
'I hoped I'd be able to have an apartment in town. But now I will live with my parents while I go to university.
The auction was hit by controversy three weeks before its culmination when a teacher at Alina's former school claimed she was not a virgin.
But Alina, who had already undergone a medical examination, was seen by a second doctor, who confirmed at a press conference she'd never had sex.
'I hoped I'd meet a nice man, like in the film Pretty Woman,' Alina said. The final bid of £8,800 was short of the £50,000 she needs to pay for her education, but Alina said the experience was not unpleasant
Alina promised in her auction pitch that she would bring documentation proving she was a virgin, and said she would agree to forgo the use of condoms on the condition the winning bidder provided her with certificates proving he was free from STIs.
teenager, from Caracal in Olt country, posted an advert on German
dating website www.gesext.de, saying: 'I am a 108lbs, 5ft 6in tall,
brown-eyed Romanian girl.
'I don't smoke and own a certificate from a gynaecologist which says I'm a virgin. I want my first time to be special and not very abrupt.
'I want to meet a gentle, respectful and generous man.'
It wasn't just sex that Alina was after either. Although her first concern was to raise money for her education, Alina was open to finding more from the transaction, saying that if she should meet her 'happiness and future, then that would be great too'.
And could it be that Alina has found love after all?
'I hope to see the man again,' she says. 'And next time I won't make him pay!'
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subset: commoncrawl


text: On Fri, 2008-06-27 at 20:27 -0700, Joel Becker wrote: > On Fri, Jun 27, 2008 at 10:06:01AM -0500, David Teigland wrote: > > The hope that I do still hold out for using logsys, is that we may be able > > to develop a simple syslog-like api for it, that would either parallel or > > sit above the plugin-oriented api that's there now. Steve claims that the > > syslog(3) api is fundamentally broken and didn't sound at all interested > > in the idea, but maybe others are more open to the idea? > > I wonder what's fundamentally broken about it. I'm sorry that > logsys doesn't use syslog internally - how does it log remotely, for > example? :-) > > Joel > The syslog api has served thousands of applications for 20+ years. But the fact is every single application that is significant develops their own logging solution because, frankly, the syslog API is a POS. The logsys API does use the syslogd daemon's ipc connection but not the c library api which is fundamentally flawed. As a result we support all of the syslogd features that system administrators have come to know and love, and doesn't change any of those features at all! For logging remotely, all of that is managed by syslogd. Syslogd is a great system. My sole issue is with the c library api syslog/openlog/closelog friends. They were designed in 1970's and are missing some key features. This is the motivation behind logsys. LOGSYS IS A REPLACEMENT for SYSLOG(3) NOT SYSLOGD. It is 100% compatabile with the syslogd daemon shipped in every single distribution in the world. The logsys api is not "plugin oriented"; it is designed for large software projects that have several components each of which may desire to log specific information at different levels, to different files, or with different tracing levels about different subsystems. Also the syslog api itself is broken because is does not allow nonblocking IO without the loss of log messages. None of this is supported by the syslog api which is why it is insufficient. But I can tell I'm talking to a BRICK WALL now since I've been repeating this for 6 months or more, and after everyone seemly agrees logsys is the way to roll, NOW IT IS CRITICAL TO REWRITE THE API because it uses a macro to support subsystems. I try not to take personal insult when these sorts of things happen but seriously, the api has been published for over 6 months and now it is not sufficient? Where were these posts 6 months ago? IMO the naysayers are a day late and a dollar short. sigh -steve


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Here are the judging results of Thursday's market hog competition at the Washington Fair.
There were 152 animals judged Thursday ranging in weight from 240 to 290 pounds. They were divided into 10 weight classes for judging.
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Translation of "rook" - English-Spanish dictionary
(Translation of "rook" from the PASSWORD English-Spanish Dictionary © 2014 K Dictionaries Ltd)
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the activity of knitting something, or a thing that is being knitted


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WTB Women's Deva Race SE Saddle - Performance ExclusivePerfect for road or cross-country racing, the WTB Women's Deva Race Special Edition Saddle has strategic padding and gel inserts to dissipate vibrations. Flex-Tuned nylon shell achieves the ideal blend of firm support and flex to enhance pedaling efficiency. WTB's proprietary Love Channel and Comfort Zone relieve pressure on sensitive tissues. Soft DNA padding and gel inserts are carefully placed to maximize comfort on longer rides. Exclusively available at Performance Bicycle.
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I901 Ind Co2 Replacement Kit - Sospenders
This item is found in the following Product Families
Includes one cylinder and one pin.
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Rent Handbook of Injury and Violence Prevention 1st edition today, or search our site for other textbooks by Lynda Shane Doll. Every textbook comes with a 21-day "Any Reason" guarantee. Published by Springer.
Need help ASAP? We have you covered with 24/7 instant online tutoring. Connect with one of our tutors now.


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St. Petersburg - Clearwater Hotel Guide
Enjoy Smiles and Sunshine in St. Petersburg
The Sunshine City definitely earns its reputation for being a lively, artsy, and eccentric place. In fact, prepare to find a healthy mix of high-rise buildings and quaint galleries dotting the landscape here. St. Petersburg's downtown is consistently ranked among the best in the aouthern United States and hosts a number of fanciful festivals throughout the year. The city's layout makes it essential to rent a car in St. Petersburg Clearwater to get to top attractions without unnecessary hassles.
A Climate of Sunshine
You already know about St. Petersburg's reputation for endless sunshine. Did you know that the city also enjoys consistently warm temperatures to accompany all those clear skies? It's not uncommon for winter days to reach 22 degrees Celsius while summertime temperatures tend to linger around 32 degrees Celsius. Whether you're escaping the chill or simply looking to chill out in the sun, relaxing at St. Petersburg Florida hotels is always a great decision.
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Are you looking for fun things to do in St. Petersburg? We recommend visiting the Salvador Dali Museum, Tropicana Field, and the St. Petersburg Pier. Spa Beach and North Shore Beach are the two stunning stretches of sand to discover along the city's waterfront area. You can rent paddle boats, hop on a toboggan slide, or simply enjoy velvety white sand. And don't run out of sunscreen before you can get to Fort De Soto Park, St. Petersburg Municipal Park, and Maximo Park. If you're worried about wearing out your wallet before you're done planning your stay here, count on Expedia to deliver amazing discounts for all your travel needs.
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"If you establish a democracy, you must in due time reap the fruits of a democracy. You will in due season have great impatience of the public burdens, combined in due season with great increase of the public expenditure. You will in due season have wars entered into from passion and not from reason; and you will in due season submit to peace ignominiously sought and ignominiously obtained, which will diminish your authority and perhaps endanger your independence. You will in due season find your property is less valueable, and your freedom less complete." ~ Benjamin Disraeli
Borderline Case: Some Real News Beyond the "Reset" Rhetoric
Submitted by Jad Davis on Mon, 2011-05-30 00:00
"Of course, anyone with the slightest acquaintance of history (which, of course, leaves out 97 percent of the Anglo-American chattering classes) knows that the United States has always been firmly and forthrightly committed to democracy for all god's chillums all over the world – as long as they vote for the leaders that Washington wants."
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Coding Freedom
Coding Freedom book release event (Friday, November 16)
Hacker culture scholar Gabriella Coleman writes:
- I will be in San Francisco for the American Anthropological Association meetings two weeks after the publication of my first book on the politics of free software: Coding Freedom: The Ethics and Aesthetics of Hacking (http://press.princeton.edu/titles/9883.html). It will be released with a CC license.
- I did a chunk of my fieldwork in San Francisco between 2001-2003 and thought it would be quite fitting to hold a book event while visiting the foggy city and Noisebridge came up as a possible venue.
- It would be amazing to hold an event in the Mission, at a hacker space, after the culmination of such a LONG project where I can also thank those, like Seth Schoen, who is not only featured in the book but also was a great friend during my time there (and still is, just now remotely).
- I am thinking of holding the event on Friday November 16. I can provide snacks and drinks and can assure you that if held, it would only attract modest numbers; I imagine a handful of Anthropologists, folks from the EFF, some local open source developers, and perhaps a few interested stragglers might show up.
Not all events make it onto this calendar. Many events only make it to the Discussion or Announcements mailing lists, IRC or in person at Tuesday meetings. Best of all, Noisebridge is about people getting together at the space in San Francisco to do stuff. DO pay attention, as some events just arise organically from the bottom up!
Want to host your event at Noisebridge? We like seeing classes and talks on interesting things pertaining to various subjects of hacking. Most of all, we like seeing familiar faces. Please participate in the space and our weekly Tuesday meetings to see if we're the right audience for what you want to share before announcing a new event. Additionally, please see our events hosting page for suggestions on how to use Noisebridge for your event/class/workshop.
Upcoming Events edit
Basic Income Wikipedia Jam,
This will be a small gathering of folks working together to clean up the Wikipedia page for "basic income," which is a total mess right now and the first resource many people new to the idea will encounter. There are neutrality issues, citations missing, key events unmentioned, overly verbose writing, confusing organization: on and on and on. If you're into the idea of basic income, come lend a hand. We'll have snacks! Wednesday, July 27, 18:30:
Post-quantum Crypto Talk by Peter Schwabe,
In 2012, Mark Ketchen, researcher at IBM, stated about large quantum computers that they are "within reach" and estimated a timespan of 10 to 15 years until such computers can be built. It is not clear if Ketchen is right with this estimate, it is not even clear if a large quantum computer will ever be built. However, what is clear is that such a computer will be able to break all asymmetric cryptography in wide use today. More specifically, it will break in polynomial time systems that are based on factoring (like RSA) and systems based on the discrete logarithm (like DSA, and Diffie-Hellman key exchange), including their elliptic-curve variants. There are asymmetric cryptographic systems that, as far as we know, are not broken by quantum computers, so called "post-quantum cryptography". It is obvious that once large quantum computers exist, the world will need to switch to such post-quantum schemes. However, users who are concerned about long-term security, have to switch to post-quantum schemes for confidentiality already now: an attacker who records and stores key exchanges today can go back in a decade or two and use a quantum computer to attack them. In my talk I will give a brief overview of post-quantum crypto and then highlight what we can, and should, already do today to provide long-term security in cryptographic systems. In particular, I will present the "NewHope" key exchange, which is currently used in an experiment by Google and is one of the candidates to be considered for post-quantum key exchange in Tor. Thursday, August 4, 12:00:
The 13-Aug GODWAFFLE NOISE PANCAKES has Noisebands and gourmet vegan pancakes!
Bands: Medicine Cabinet (Tracy CA), Daniel Blomquist, Ze Bib!, Foreskin Shashimi in Paradise, SubStation6' Saturday, August 13, 12:00:
TOOOL SF Bay Area Lockpicking Meeting, Sunday, August 21, 13:00:
The 10-Sep GODWAFFLE NOISE PANCAKES has Noisebands and gourmet vegan pancakes!
Bands: Rick & Joe Potts (LAFMS), Voicehandler, Gif Problem, Tender Buttons, Rust Worship Saturday, September 10, 12:00:
The 8-Oct GODWAFFLE NOISE PANCAKES has Noisebands and gourmet vegan pancakes!
Bands: John Bischoff, Acutuary (L.A.), Striations, Article Collection (Boston), The Flayed Choirmaster Saturday, October 8, 12:00:
Recurring Events edit
E - Every week.
1st - Certain weeks, e.g. 1st week only.
-2nd - Except certain weeks, or other schedule exceptions.
- E 7:00 pm to 9:00 pm Circuit Hacking Mondays (Early start of 3:00pm on Monday holidays) - Learn to solder! And make cool things with electronics. Mitch, J, Rolf, Miloh, Cedric, Cheng, Greg, and/or a host of others will bring kits-for-purchase to make cool, hackable things for all skill levels that you can bring home after you make them! Many designed for absolute beginners! Bring your own projects to hack! Bring things to fix! All ages. All are welcome! See the Discuss list and the Announce list for weekly updates.
- E 8:00 pm to 10:00 pm Front-end Web Development - Learn HTML/CSS/JS. A series of talks on different topics every week, taught in the Church classroom. Recap of last week's material starts at 7:30 pm. Join the WebDev list or the Announce list for updates.
- E Take Out the Trash Night -- Round One Be sure to put bins out by midnight as the trash truck comes between 1:30 to 2:30am.
- E 7:00 pm to 9:00 pm Intermediate Python with PyClass - Crash course in the Python standard library. Monday session is held in Turing Classroom.
Super Tuesdays!
- E 5:00 pm to 7:30 pm Songbridge Music Making Tuesdays - Beginner-friendly music making and mentoring meetup with Ableton/GarageBand/Logic tutorials for beginners and peer collabs. (Noise Square Table).
- 1st 3rd 6:30 pm to 8:30 pm Sewing and Crafting - Come learn how to use the sewing machines or just work on a craft (e.g. knitting, crocheting, felt working, embroidery, leatherworking, beading, etc.). All skill levels are welcome.
- E 7:00 pm to 9:00 pm Ruby and Rails class - Seminar and workshop for learning everything about Ruby, Rails, and web application development (Church classroom). See the Announce list for weekly updates.
- E 8:30 pm to 11:30 pm Gamebridge Unityversity Game Dev Tuesdays - Beginner-friendly game development class and meetup, learn and share C# Unity coding, 2D/3D art, design, writing and audio. Learn how to mentor and help teach game dev. (Noise Square Table).
- E 8:00 pm Noisebridge Weekly Meeting - Introducing new people and events to the space, general discussion, and decision-making on key issues. This is your space, folks. Come on out here in person to express what you think about what's going on with it!
- alternating monthly 6:30 pm to 9:30 pm - Cyberspectrum: Software Defined Radio Meetup in the Hackatorium(new): A place to learn and exchange ideas about SDR. Presentations on concepts, mystery signals, hardware/software and cool applications. Event alternates monthly between SF and the South Bay.
- 1st Wednesday of every month. Art with Software 7:30pm. Present your personal and expressive art made with software/hardware of your own making and get feedback and critical dialogue from fellow artist/technologists. More info on Meetup webpage here. [Last upded July, 2016].
- E 8:00 pm to 10:00 pm Dream Team Neuro Hackery - EEG research & development project with general interest in sleep, dreaming, creative intelligence, and many loosely related topics such as: neurophysiology, signal processing, cognitive neuroscience, and (especially) hacking code and devices for data acquisition and analysis. Join us at the CollaborationStation (near the Hackatorium). Expect general discussion around 8 PM - usually moving along by 9 PM to focus on more technical aspects of current project.
- E Wednesday (3/23/16) CANCELLED for Spring Break SFSU resuming (3/30/2016) 8:30 pm to 10:30 pm, aka 20:30-22:30, class times, Just add You! Raspberry Pi - NEW PiBridge Raspberry Pi Hacking Group is a meetup to explore on Raspberry Pi-based projects. Gain momentum and achiving successes! '. Meeting lateral to the Pi Zero Student Learning Station, in the Hackatorium Class Space.
- E Trash Night -- Round Two - Take out the trash for Friday morning! Do it by midnight as truck comes ~1:30am to 2:30am.
- E 7:00 pm to 9:00 pm Front-end Web Development Lab - Understand by doing! A recap of Monday's lecture in workshop form - and a good time for one-on-one help with the material. Meets in Turing.
- 2nd 7:00pm to 9:00pm Cryptopal working group - Learn cryptography through coding challenges that demonstrate attacks on real-world cryptography. A different way to learn than taking a class or reading a book!
- 3rd 7:00 pm Five Minutes of Fame a.k.a. 5MoF - lightning 5min talks every 3rd Thursday of the month
- 1st 3rd 5th 6:00 pm to 8:00 pm Computerology in Turing or CollaborationStation, understanding and using computers
- 1st 3rd 5th 8:00 pm to 10:00 pm FUN Tutoring @ CollaborationStation
- 3rd Noisebridge Gaming Archivists Meetup 6:00 pm to 10:00 pm -- Come play, fix, mod retro consoles. Create and play is our motto! If you like video games, this is the place for you.
- E 1:00 pm to 2:00 pm CPP Class discussion in Hackitorum/Fox Lounge/Church. Currently looking for folks wanting to help plan out lessons.
- E 1:00 pm Zine Hacking Hacking on the good ol' fashioned analog Zine with Torrie, Mari and anyone who wants to come and be excellent and make excellent art.
- alternating monthly 3:00 pm Goths & Crafts Spooky people making spooky things in the sewing area. All kinds of crafts welcome and things don't necessarily have to be goth. But if you have an eye towards the darker side of the color spectrum, this may be for you!
- E 12:30 pm to 7:30 pm Dungeons and Dragons in Church, currently looking for new players.
- alternating monthly 1:00 pm Monthly Lock Sport Collaboration: Come learn how to pick locks and learn more about them with the SF Bay Area chapter of TOOOL. The group meets on alternating months at Noisebridge and in San Jose. Check the TOOOL SF announcement list for details.
- E 3:00 pm Go - Playing of the Go board game. On nice days we often take the boards to Dolores Park and play there.
- E 6:00 pm Plan 9 class (if not Noisebridge check Sycamore)
- E 12:00 pm to 4:00 pm .impact Workathons in Turing classrom. Work on projects that will help humanity & beyond.


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7 July 2014
European private equity firm Doughty Hanson has doubled its initial investment in Spanish private hospital group Quirón by selling its stake to CVC Capital Partners.
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Stay 5 / Pay 4 (S5P4) [Valid from 01 May 2016 to 31 Mar 2017]
Treasure Island ResortLocation: Mamanuca Islands
Set on a coral and sand-fringed coral cay 17 km from Nadi airport in the famous Mamanuca Islands, Fjii. Treasure Island is one of Fiji's most popular resorts. Designed for honeymooners, couples and families, the property offers 66 renovated beachfront bures positioned for privacy and views. This Fijian resort boasts a three-pool complex, refurbished restaurant-bar, and a Kids' Club bure. The resort also features a stunning wedding chapel and a variety of leisure and cultural activities.
[more info]
50% Discount (TAG50) [Valid from 12 May 2016 to 31 Mar 2017]
(AUG40) - 40% Discount on Accommodation - minimum 4 night stay - All Categories [Valid from 01 Aug 2016 to 31 Aug 2016]
*'SEPTEMBER SCHOOL HOLIDAY BLITZ' *30% Discount [Valid from 19 Sep 2016 to 10 Oct 2016]
Matava ResortLocation: Kadavu
Matava Resort offers eco-friendly accommodation providing privacy, comfort and ocean views out over the Great Astrolabe Barrier Reef.
7 Nights/10 Tanks Package [Valid from 01 Apr 2016 to 31 Mar 2017]
6 Nights/10 Tanks Dive Package [Valid from 01 Apr 2016 to 31 Mar 2017]
6 Nights/10 Tanks Dive Package [Valid from 01 Apr 2016 to 31 Mar 2017]
5 Nights/8 Tanks Dive Package [Valid from 01 Apr 2016 to 31 Mar 2017]
Taveuni PalmsLocation: Taveuni
The boutique resort of Taveuni Palms rests along a white sandy beach with spectacular views of the ocean from the private villa accommodations. Taveuni Palms is ideal for romantics wishing to escape to a secluded retreat and enjoy the warm weather, stunning snorkelling and diving adventures, gourmet cuisine and the friendly service of the local staff.[more info]
STAY 5 PAY 4 (TP5/4) [Valid from 21 Jun 2016 to 31 Oct 2016]
STAY 5 PAY 4 - TP5/4 [Valid from 01 Aug 2016 to 31 Oct 2016]
Navutu Stars Resort FijiLocation: Yasawa Islands
Navutu Stars Resort is a small boutique resort on the island of Yaqeta in the Yasawa region.
Inclusions: full gourmet continental breakfast, Meet and Greet at Airport, welcome cocktail at resort on arrival and departure, fresh tropical fruit platter in bure on day of arrival, 30 minute arrival relaxation massage using organic treatments in the Navutu Spa (per guest per stay). Conditions apply. Visit the price page to view an up-to-date list of inclusions, free activities and services.[more info]
Stay 5 Pay 4 OR Stay 7 Pay 5 OR Stay 10 Pay 8 [Valid from 01 Apr 2016 to 31 Mar 2017]
Likuliku Lagoon ResortLocation: Mamanuca Islands
Likuliku Lagoon Resort rests on 800 metres of pristine palm fringed beachfront with a number of the accommodations located over the turquoise waters. A romantic retreat for adults only, you easily lose track of time as you relax by the freshwater pool or under the shade of a palm tree. You can enjoy snorkelling in clear waters only steps away from your bure. Rejuvenate your body and soul at the Tatadra spa. Indulge in the exquisite flavours of Fiji and the Pacific Rim.[more info]
"Indulgence Package" [Valid from 01 Apr 2016 to 18 Dec 2016]
"Celebration Package" [Valid from 01 Apr 2016 to 31 Mar 2017]
"Short Break Package" [Valid from 01 Apr 2016 to 31 Mar 2017]
Low Season Bonus [Valid from 01 Nov 2016 to 31 Mar 2017]
Radisson Blu Resort Fiji is Denarau Island's newest 5 star luxury beachfront resort.Offering a myraid of resort facilities nestled around lagoon swimming pools and lush tropical gardens combined with warm genuine Fijian hospitality makes Radisson Blu Resort Fiji Denarau Island is the perfect choice for pure relaxation, a family getaway or your next event.
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RADISSON BLU - RIGHT HERE RIGHT NOW [Valid from 16 Jul 2016 to 10 Oct 2016]
Tropica Island ResortLocation: Mamanuca Islands
Tropica Island Resort is located on Malolo Island in the stunning Mamanuca group of islands.[more info]
Stay 5 / Pay 4 [Valid from 01 Jul 2016 to 31 Mar 2017]


text:
|Publication number
|Publication type
|Application number
|Publication date
|May 13, 2008
|Filing date
|Apr 25, 2005
|Priority date
|Apr 23, 2004
|Fee status
|Also published as
|Publication number
|Tobin Hellyer, Thomas Fort, Ray A. McMillian
|Original Assignee
|Becton, Dickinson And Company
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (16), Non-Patent Citations (12), Referenced by (5), Classifications (8), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
Priority is claimed under 35 U.S.C. � 119(e) to provisional application U.S. 60/564,926, filed Apr. 23, 2004.
The present invention relates to a method of determining the efficacy of an extraction step in a process for the working up of a biological sample containing a nucleic acid. The present invention also relates to a packaged array for extracting a nucleic acid from a biological sample.
Extracting a nucleic acid from a sample is an important operation in processes in clinical diagnosis, cloning, purification and isolation and other processes in biotechnology. For instance, gene recombinant technology requires the isolation of both a vector DNA and the cloned and/or expressed DNA. In order to diagnose a genetic disease or detect a cancer gene, it is necessary to extract the desired nucleic acid from the tissue, the cells, and the various other biological materials in a sample.
A nucleic acid does not occur free in nature. It is found in bacteria, cells, or virus particles, surrounded by a cell membrane and/or cell wall composed of proteins, lipids and sugars. A nucleic acid generally forms complexes with histone and/or other proteins in its natural environment. To extract a nucleic acid, the surrounding cell membranes and cell walls must be disrupted. In the case of isolating a nucleic acid, the nucleic acid-protein complex needs to be denatured or degraded to free the desired nucleic acid from the complex so that it can be solubilized and extracted. Methods of extracting nucleic acids are described, for example, in U.S. Pat. No. 6,043,032, incorporated herein in its entirety.
Internal standards have been applied in nucleic acid analysis. These include constitutively expressed mRNAs to control for the effectiveness of the workup. Further, external controls have been applied in the extraction step in nucleic acid amplification-based analysis. However, the detection and/or quantification of the control has required amplification, so it is not possible to distinguish whether a problem in the process has arisen from the amplification step or in the extraction step. For example, in U.S. Pat. No. 6,387,652 B1, addition of G. candidum sequences as a reference to assays for fungal target sequences was employed as a control. However, it was necessary to assume an amplification efficiency of one (col. 19, lines 45-61).
Up to the present there has been no method of verifying the extraction process alone for a nucleic acid, independent of the downstream application, such as an amplification step.
The present invention relates to a method of determining the effectiveness of an extraction step in the workup of a sample of a target nucleic acid. The present invention further relates to a packaged array for extracting a target nucleic acid from a biological sample. In a further embodiment, the method and packaged array of the present invention can be incorporated in a robotic assembly for the automated analysis for the presence of a disease organism in a biological sample.
The present invention relates to a method of ensuring or verifying the extraction of a target nucleic acid from a biological sample.
The target is a nucleic acid such as single or double-stranded DNA and RNA. Examples of nucleic acid that can be extracted by the method include not only genomic DNA or RNA from animals, plants, bacteria, viruses, fungi and parasitic organisms, but also the DNA or RNA of mitochondria or chloroplasts. Examples of other classes of nucleic acid that can be extracted by the method include not only mRNA, but also tRNA, rRNA, and tmRNA (transfer-messenger RNA) as well as plasmid DNA. DNA and RNA extracted by the method of the invention may also be either wholly or partially single-stranded or possess other tertiary or quaternary structure. A sample containing nucleic acids is exemplified by viable samples such as leukocyte cells, the culture of host cells containing vectors or the like that are typically prepared by gene recombinant technology, cells infected with viruses or phages, viruses in blood, and the culture of a sample microorganism. The culture may contain microorganisms but its supernatant alone is sufficient. Not only an artificial culture but also a naturally occurring culture is applicable. In case of a sample containing lumps of microorganism, homogenization or sonication may be performed as required to achieve good efficiency of extraction.
Alternative sample types include but are not limited to biological specimens for the diagnosis of infectious or non-infectious diseases, environmental specimens, or samples of food or water. The target nucleic acid may be a particular sequence or it may be a class of nucleic acid. A class of nucleic acid is, for a particular assay method, those molecules of nucleic acid whose chemical, physical or biological properties are such that they can be expected to be extracted effectively in methods used for nucleic acid extraction. Typically, but not necessarily, the nucleic acids of a class are all DNA or DNA analogs or all RNA or RNA analogs.
Targeted organisms can include but are not limited to Chlamydia trachomatis, Neisseria gonorrhoeae, Human Immunodeficiency Virus � , Hepatitis C Virus, Hepatitis B Virus, Severe Acute Respiratory Syndrome Virus, Influenza A/B, Herpes Simplex Viruses 1-6, Enteroviruses, West Nile Virus, Parainfluenza viruses, Adenoviruses, Respiratory Syncytial Virus A/B, Mycobacterium paratuberculosis, Mycobacterium avium-intracellulare complex, Mycobacterium tuberculosis complex, Cytomegalovirus, Group B Streptococcus, Bordetella pertussis, and Bordetella parapertussis.
In one aspect of the invention, the target nucleic acid is a particular RNA or cDNA from one ore more of the following sources: bacterial pathogens, bacterial non-pathogens, viral pathogens, viral non-pathogens, fungal pathogens, fungal non-pathogens, yeast pathogens, yeast non-pathogens, parasitic pathogens, parasitic non-pathogens, plants, animal products, food, total RNA or cDNA within the sample matrix, total prokaryotic RNA or cDNA, total eukaryotic RNA or cDNA, or total viral RNA or cDNA.
In another aspect of the invention, the target nucleic acid sought is DNA from one or more of the following sources: bacterial pathogens, bacterial non-pathogens, viral pathogens, viral non-pathogens, fungal pathogens, fungal non-pathogens, yeast pathogens, yeast non-pathogens, parasitic pathogens, parasitic non-pathogens, plants, animal products, food, total DNA within the sample matrix, total genomic prokaryotic DNA, total genomic eukaryotic DNA, or total viral DNA.
According to the method of the present invention, an extraction control is added to the biological sample before the extraction step. The extraction control is a nucleic acid sequence with a distinguishable label. By distinguishable label is meant a label or marker that can be identified and quantified by a radiometric, spectrometric, fluorogenic or colorimetric method in the presence of unlabeled nucleic acid and other components of the sample matrix. The use of an extraction control for the extraction process is understood as the use of a reference or standard to allow for verification that the extraction occurred as desired. Calibration and quantification of the extraction control are also possible. The extraction control is designed to be detected without amplification of its nucleotide sequence, and further designed so as not to interfere with amplification of the target nucleic acid.
Nucleic acid labels are known in the art. These include, but are not limited to, donor quencher dye pairs such as fluorescein isothiocyanate (FITC)/tetramethylrhodamine isothiocyanate (TRITC), FITC/Texas Red™ (Molecular Probes), FITC/N-hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC), N-hydroxysuccinimidyl 1-pyrenesulfonate (PYS)/FITC, FITC/Rhodamine X, FITC/tetramethylrhodamine (TAMRA), and others. The selection of a particular donor/quencher pair is not critical. For energy transfer quenching mechanisms it is only necessary that the emission wavelengths of the donor fluorophore overlap the excitation wavelengths of the quencher, i.e., there must be sufficient spectral overlap between the two dyes to allow efficient energy transfer, charge transfer or fluorescence quenching. P-(dimethyl aminophenylazo) benzoic acid (DABCYL) is a non-fluorescent quencher dye which effectively quenches fluorescence from an adjacent fluorophore, e.g., fluorescein or 5-(2′-aminoethyl) aminonaphthalene (EDANS). Any dye pair which produces fluorescence quenching can be used in the methods of the invention, regardless of the mechanism by which quenching occurs.
Preferred labels for extraction control include fluorophores such as fluorescein and rhodamine, radioactive labels such as 32P or 35S, enzymes such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, soybean peroxidase or luciferase. Methods for linking the detectable moieties with the nucleic acid in a form that is stable to most methods of handling nucleic acid are known in the art. For example, methods for making covalent linkages are provided commercially by companies such as Integrated DNA Technologies (see IDT technical bulletins, available via the internet at the Integrated DNA Technologies website). Other labeling methods include having the detectable moiety attached to one member of a highly stable binding pair, and the other member of the binding pair attached to the nucleic acid. Such a binding pair can be, for example, avidin (or streptavidin) and biotin. Avidin-biotin labeling techniques are described for example in Advances in Biomagnetic Separation (M. Uhlen, E. Hornse, and O. Olsvik) (Eds.), Eaton Publishing 1994).
The nucleotide sequence of the extraction control must be sufficiently long to have the general physical and chemical properties of nucleic acid. In one embodiment, the extraction control consists essentially of a nucleotide sequence between 21 and 61 bases in length and a label. In another embodiment, the nucleotide sequence, labeled with for example rhodamine, must be sufficiently long to reversibly bind to an iron oxide particle under extraction conditions such as described in U.S. Pat. No. 6,433,160, where free rhodamine does not bind to an iron oxide particle.
The extraction control can be added to a biological sample before or after lysis/disruption of the cell membrane or cell wall. In certain circumstances, such as with an RNA-based extraction control that is susceptible to degradation by RNAses, the extraction control should not be added until the point immediately before extraction. For procedures involving binding of nucleic acid to a solid support, the extraction control generally may be added at any point prior to the binding, and thereby control for all subsequent phases of the process.
Following addition of an extraction control, the step of extracting nucleic acid is performed. The phrase "extracting nucleic acid" refers to purifying nucleic acid sufficiently away from protein or other material within the sample matrix so that it has purity reasonably sufficient for assays to identify or quantify segments of nucleic acid. The term "protein" is used to include chains of amino acids or amino acid derivatives comprising peptides, polypeptides or full-length proteins. Methods of extracting nucleic acids are known in the art. For example, U.S. Pat. No. 6,043,032 describes several methods of extracting nucleic acid, including liquid phase extraction methods, incorporated herein in its entirety.
The extraction procedure can include a step in which the nucleic acid is bound to a solid support. The solid support is typically washed to remove undesired material. In many instances, the nucleic acid is released from the solid support and processed further. In other instances, the nucleic acid bound to the solid support is processed further. Those of skill will recognize when it is appropriate to assess extraction as opposed to assessing a subsequent procedure. Often, the assessment is made after release from the solid support. Or, if no such release is required, the assessment can be made of the nucleic acid bound to the support. Appropriate supports for non-specific capture of nucleic acids include, but are not limited to, crushed glass powders (e.g., available from Bio101 as GeneClean�) and glass fiber filters (e.g., available from Roche as the High Pure™ system, Celite (e.g., available from BioRad Laboratories as Prep-A-Gene™), and iron-containing paramagnetic particles (e.g., as described in U.S. Pat. No. 6,433,160, which discloses nucleic acid binding under acidic conditions). For target-specific capture, paramagnetic particles with modified surfaces may be used (e.g., such as described in European Specification EP 0 446 260 B1 and U.S. Pat. No. 5,512,439, with each particle carrying a plurality of molecules of an oligonucleotide). The captured oligonucleotides may be conjugated directly to the particle surface or coupled via an intermediary linker such as a streptavidin-biotin or other receptor-ligand interaction, as is known in the art.
Following extraction to yield an extract containing the extraction control and the target nucleic acid, the presence of the extraction control in the extract is verified by detection of the label, also known as a detectable marker. The extraction control label can be further analyzed by the detection method to allow for quantification of the amount of extraction control in the extract. Suitable methods for detection of the label include radiometric, spectrometric, fluorogenic or colorimetric methods.
The extraction control can have a detectable marker whose detection characteristics (e.g., emission or absorption wavelengths) overlap the detection characteristics that will be utilized in a post-extraction procedure, such as an assay for a specific molecule. For example, the chromophores or fluorophors used for the extraction control and in the second post-extraction procedure can be the same. In such case, the detection of the extraction control and the detection in the subsequent procedure are designed so that a positive extraction yields an amount of detectable marker that will, in the subsequent procedure, contribute only an amount of signal to the result as can be reliably compensated for in a controlled-for background signal.
The data from detection of the labeled extraction control is optionally normalized. The step of normalizing data can comprise interpolating from the control data how much any experimental data point should be normalized or can comprise discarding data points that the control data indicates is unreliable or which may be considered as background signal. The detection and normalization of data can be combined in a single step for purposes of automation.
In one embodiment, the nucleic acid sequence of the extraction control is substantially based on the structure of the target nucleic acid. Thus, for example, the method can seek to extract a given structural gene, open reading frame (ORF), intron, exon, mRNA, cDNA, and the nucleic acid of the extraction control can be designed to contain 1% or more, 10% or more, or 50% or more of the primary structure of the same. Thus, in one embodiment, if RNA is sought to be extracted, the extraction control will be RNA (or an RNA analog), and if DNA is sought to be extracted the extraction control will be DNA (or a DNA analog). In other embodiments, if RNA is to extracted, the extraction control may be DNA (or a DNA analog) and if DNA is to be extracted, the extraction control may be RNA (or an RNA analog).
A structural gene is defined by the nucleic acid segment (and if relevant its complement) that codes for a transcribed RNA (whether such RNA is later edited to remove introns or the like) or that codes for the minimum contiguous segment that codes for an expressed protein. Given the existence of splice variants, and the typical existence of RNA at the 5′ and 3′ ends of mRNA that is not translated into protein, a given gene may define one, two or several structural genes. In all cases, the presence of a modification with a label of a residue or nucleotide, which would otherwise comprise shared sequence, does not diminish the shared percentage. For recitations of percent identical structure, if the percentage required exists for at least one of the structural genes, then the recitation is satisfied.
In another embodiment, if two or more target nucleic acids with different characteristics are to be extracted, two or more extraction controls may be added before the extraction step.
If the target nucleic acid to be extracted has features that are associated with difficulties in extraction, these features may be modeled in the substances selected as the extraction controls. For example, if a particular nucleic acid segment is sought, and that segment includes a high G/C content segment, or a high degree of secondary or tertiary structure that contributes to the difficulty of extracting the desired segment, then such sub-segment or a homolog thereof may be included in the sequence of the extraction control. The design of the extraction control also includes the limitation that the extraction control should not interfere with post-extraction amplification of the target sequence.
In many contexts for extracting nucleic acid, a hybridization reaction will be used in a post-extraction procedure. For many such post-extraction procedures, the use of nucleic acids for the extraction control with a sequence based on an intron sequence will minimize undesired competing hybridizations. Internal hairpin structures can also minimize undesired competing hybridizations. Where polymerase-based methods are used in post-extraction procedures, the 3′ end of the extraction control can be modified to prevent extension of the extraction control. For example, the detectable marker can be attached to the 3′ end to block extension or the 3′ end of the extraction control may be capped using a dideoxynucleotide, inverted base or other non-extendable terminal moiety as is known in the art. Other methods of inhibiting undesired amplification are known in the art, for example in U.S. Pat. No. 5,972,610 and U.S. Pat. No. 5,849,497. Determining whether or not an extraction control will interfere with amplification of the target sequence can be accomplished by running in parallel an amplification of the target sequence with and without the extraction control.
A distinction is made between nucleic acid amplification and signal amplification. By nucleic acid amplification is meant a technique such as SDA, PCR, TMA, NASBA, etc., whereby additional copies of the nucleic acid are made. In contrast, signal amplification such as that which occurs with a chemiluminescent or calorimetric label is used in the detection of a label, and does not result in additional copies of the label. The use of the general term "amplification" in reference to post-extraction amplification is meant to refer to nucleic acid amplification. An extraction control sequence is defined as not capable of being amplified during post-extraction amplification if, at the conclusion of amplifying the target sequence, the amount of extraction control sequence present in the reaction mixture is less than 1000-fold more than was present prior to amplification. The extraction control of the present invention is designed to not participate in or interfere with a subsequent amplification step of the target nucleic acid, including amplification of sequences added to independently measure or control for the efficiency of the amplification step. Non-participation or non-interference by the extraction control is defined by running parallel amplifications of the target sequence with and without the extraction control. If the amplification of the target sequence in the presence of the extraction control is roughly equivalent to the amplification of the target sequence without the extraction control, then the extraction control is defined as not participating, or not interfering with, the post-extraction amplification of the target sequence.
In one embodiment of the invention, multiple extraction controls are included in the extraction procedure, each designed specifically to verify the extraction of one or more classes of nucleic acid. In another embodiment of the invention, the extraction control can be dried for long-term storage without impacting its form or function.
An experiment was conducted to evaluate the ability of a Rhodamine (ROX) labeled extraction control to detect automated nucleic acid extraction failures. The control was designated BBTEC-26 (SEQUENCE ID NO. 1). The protocol was as follows:
The data below are presented in MOTA values for the CT determinations and in machine-normalized ROX values for the EC determinations. Free ROX dye will not bind to ferric oxide under the conditions used for the extraction. The MOTA values represent the sum of individual fluorescence measurements over time using a fluorometer with an established cutoff level for a positive reaction of 2,000 for the BD ProbeTec™ ET CT and GC Amplified DNA Assays. BD ProbeTec™ brand assay kits are available from Becton, Dickinson and Company, and are designed for use with use the BD ProbeTec™ ET System for Strand Displacement Amplification (SDA). The BD Viper™ Sample Processor automates the sample handling associated with high-volume testing using the BD ProbeTec™ ET System.
EC Score (ROX Assay
EC Score (ROX Assay
Target MOTA
Well/ROX Normalizer)
Target MOTA
Well/ROX Normalizer)
No EC
+Binding Acid
+Binding Acid
No CT
+Binding Acid
No Binding Acid
The results are presented graphically in
To determine whether the EC could be extracted from different matrices, 168 pmol of the EC was spiked into pools of clinical urine and matrix from pressed vaginal swabs. Eight assay replicates were analyzed for each sample, including a negative control lacking EC. The results are presented in
The effect of the extraction control (EC) on a separate internal amplification control (IAC) was assessed by adding different levels of the EC to a pool containing matrix from pressed vaginal swabs and extracting as described in Example 1. The resulting eluates were tested in strand displacement amplification assays for CT and GC which contained internal amplification controls. The results are presented in
A sequence-specific extraction is performed by modification of Example 1. In place of iron oxide particles, streptavidin-coated beads are used. The beads are mixed with two different biotinylated oligonucleotides, one of which is complementary to the target sequence, while the other is complementary to the extraction control sequence. The extraction control is labeled with rhodamine. Appropriate conditions for hybridization as are well known in the art are used in the extraction step. Variation in salt concentration, temperature, cosolvent, and detergent are used to vary the stringency of hybridization specificity. Following hybridization, the beads are washed under appropriate conditions to remove debris and non-hybridized material. The target nucleic acid and the extraction control are eluted under conditions of low salt, elevated temperature, or other method as known in the art. The rhodamine label of the extraction control is detected to verify the extraction process, and the level of recovered rhodamine is quantified to determine the efficiency of capture, washing, and elution. The results are expressed quantitatively or qualitatively.
Publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety in the entire portion cited as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references.
While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations in the preferred devices and methods may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims that follow.
Alternate embodiments may not have been presented for a specific portion of the invention. Some alternate embodiments may result from a different combination of described portions, or other non-described alternate embodiments may be available for a portion. This is not to be considered a disclaimer of those alternate embodiments. It is recognized that many of those non-described embodiments are within the literal scope of the following claims, and others are equivalent.
|Cited Patent
|Filing date
|Publication date
|Nov 22, 1993
|Nov 19, 1996
|The Childrens Hospital Of Philadelphia
|Method of detecting genetic deletions and mutations associated with DiGeorge syndrome, Velocardiofacial syndrome, charge association, conotruncal cardiac defect, and cleft palate and probes useful therefor
|Sep 26, 1997
|Nov 17, 1998
|Akzo Nobel N.V.
|Nucleic acid quantitation by co-amplification of target with multiple internal controls
|Jul 24, 1997
|Dec 14, 1999
|Univera Pharmaceuticals, Inc.
|Method of identifying Aloe using PCR
|Nov 9, 2000
|Apr 30, 2002
|Nanogen, Inc.
|Methods for gene expression monitoring on electronic microarrays
|Jul 19, 2000
|Nov 4, 2003
|V.I. Technologies, Inc.
|Nucleic acids for detecting parvovirus and methods of using same
|Nov 2, 2000
|Nov 18, 2003
|Xtrana, Inc.
|Self-contained device integrating nucleic acid extraction, amplification and detection
|May 25, 2001
|Nov 16, 2004
|Maine Molecular Quality Controls, Inc.
|Compositions and methods relating to nucleic acid reference standards
|Dec 22, 1999
|Jun 6, 2002
|Gerard Gundling
|Nucleic acid isolation method & kit
|Dec 4, 2001
|Jun 12, 2003
|Maurice Exner
|Oligonucleotides and methods for detecting borrelia burgdorferi
|Nov 4, 2002
|Jan 20, 2005
|Hiillebrand Timo
|Reaction chambers containing complex stable reagent formulations and test kit for detection and isolation of pathogenic microbial nucleic acids
|Sep 22, 2004
|Jun 16, 2005
|Cho-Yau Yeung
|G+C-rich binding protein
|Jul 8, 2004
|Oct 27, 2005
|Collis Matthew P
|Extraction of nucleic acids using small diameter magnetically-responsive particles
|Feb 9, 2004
|Dec 22, 2005
|Diagnocure Inc.
|Method to detect prostate cancer in a sample
|May 10, 2006
|Sep 28, 2006
|Volkin David B
|DNA vaccine formulations
|Dec 15, 2003
|Jan 18, 2007
|Infectio Diagnostic (I.D.I.), Inc.
|Biological reagents and methods to verify the efficiency of sample preparation and nucleic acid amplification and/or detection
|Dec 20, 2000
|Jun 28, 2001
|Baxter Aktiengesellschaft
|Methods for the preparation and use of internal standards for nucleic acid amplification assays
|Boom R, et al., "Rapid and Simple Method for Purification of Nucleic Acids." Journal of Clinical Microbiology, Washington, DC, USA, vol. 28, No. 3, Mar. 1, 1990, pp. 495-503.
|Chelly et al., Transcription of the dystrophin gene in human muscle and non-muscle tissues. Nature 333: 858-860 (1988).
|Inglis et al., A survey of Camplobacter species shed in faeces of beef cattle using polymerase chain reaction. □□Canadian J. of Microbiology 49 : 655-661 (2003).
|Matthews et al. Review : Analytical Strategies for the use of DNA probes. Analytical Biochemistry 169 : 1-25 (1988).
|Moody A, et al., "Measuring infectious bursal disease virus RNA in blood by multiplex real-time quantitative RT-PCR." Journal of Virological Methods, Amsterdam, NL, vol. 85, No. 1-2, Mar. 2000, pp. 55-64.
|Mumy Karen L, et al., "Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR." Journal of Microbiological Methods, May 2004, vol. 57, No. 2, pp. 259-268, XP002435441 & Database Science Direct online version of document, Mar. 14, 2004.
|Petersen D G, et al., "Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGE." FEMS Microbiology Ecology, Elsevier, NL, vol. 53, No. 3, Aug. 1, 2005, pp. 339-348.
|Petersen N E, et al., "Improved RNase protection assay for quantifying LDL-receptor mRNA; estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells." Clinical Chemistry Nov. 1995, vol. 41, No. 11, pp. 1605-1613.
|Sunters et al., The use of alpha-DNA as an internal standard in the detection and quantification of DNA damage in specific genes using Southern blotting. Nucleic Acids Research 24(12) : 2456-2457 (1996).
|The Stratagene Catalog p. 39(1988).
|Tyagi et al., Molecular Beacons: Probes that fluoresce upon Hybridization. Nature Biotechnology 14 :303-308 (Mar. 1996).
|Witt D J, et al., "Techniques for the evaluation of nucleic acid amplification technology performance with specimens containing interfering substances: efficacy of boom methodology for extraction of HIV-1 RNA." Journal of Virological Methods Apr. 1999, vol. 79, No. 1, pp. 97-111.
|Citing Patent
|Filing date
|Publication date
|Apr 4, 2008
|Oct 14, 2014
|Becton, Dickinson And Company
|Use of an extraction control in a method of extracting nucleic acids
|Dec 30, 2011
|Jul 14, 2015
|Longhorn Vaccines And Diagnostics, Llc
|Compositions and methods for rapid, real-time detection of influenza a virus (H1N1) Swine 2009
|Apr 4, 2008
|Jun 11, 2009
|Becton, Dickinson And Company
|Use of an Extraction Control in a Method of Extracting Nucleic Acids
|Dec 16, 2011
|Apr 12, 2012
|Longhorn Vaccines & Diagnostics, Llc
|Biological Specimen Collection/Transport Compositions and Methods
|Dec 30, 2011
|May 10, 2012
|Longhorn Vaccines & Diagnostics, Llc
|Compositions and Methods for Rapid, Real-Time Detection of Influenza A Virus (H1N1) Swine 2009
|U.S. Classification
|435/6.11, 536/25.4
|International Classification
|Cooperative Classification
|European Classification
|Aug 8, 2005
Owner name: BECTON, DICKINSON AND COMPANY, NEW JERSEY
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HELLYER, TOBIN;FORT, THOMAS;MCMILLIAN, RAY A.;REEL/FRAME:016365/0828;SIGNING DATES FROM 20050802 TO 20050803
|Nov 14, 2011
|Fee payment
Year of fee payment: 4
|Oct 27, 2015
|Fee payment
Year of fee payment: 8


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- Magellan Behavioral Health
- McLaren
- Medicaid
- Medicare
- Meridian
- Military OneSource
- Molina
- Multiplan
- Optum
- Priority Health
- PsychCare
- United
- ValueOptions
- Addiction
- Adoption
- Alcohol Abuse
- Alzheimer's
- Anger Management
- Antisocial Personality
- Anxiety
- Asperger's Syndrome
- Autism
- Behavioral Issues
- Bipolar Disorder
- Borderline Personality
- Career Counseling
- Child or Adolescent
- Chronic Impulsivity
- Chronic Pain
- Chronic Relapse
- Codependency
- Coping Skills
- Depression
- Developmental Disorders
- Divorce
- Domestic Abuse
- Domestic Violence
- Drug Abuse
- Dual Diagnosis
- Eating Disorders
- Emotional Disturbance
- Family Conflict
- Gambling
- Grief
- Infertility
- Infidelity
- Intellectual Disability
- Internet Addiction
- Learning Disabilities
- Life Coaching
- Marital and Premarital
- Medical Detox
- Medication Management
- Men's Issues
- Narcissistic Personality
- Obesity
- Obsessive-Compulsive (OCD)
- Oppositional Defiance
- Parenting
- Peer Relationships
- Pregnancy, Prenatal, Postpartum
- Racial Identity
- Relationship Issues
- School Issues
- Self Esteem
- Self-Harming
- Sex Therapy
- Sexual Abuse
- Sexual Addiction
- Sleep or Insomnia
- Spirituality
- Sports Performance
- Substance Abuse
- Suicidal Ideation
- Teen Violence
- Testing and Evaluation
- Transgender
- Trauma and PTSD
- Traumatic Brain Injury
- Video Game Addiction
- Weight Loss
- Women's Issues
Treatment Orientation
- Acceptance and Commitment Therapy (ACT)
- Art Therapy
- Attachment-based
- Coaching
- Cognitive Behavioral (CBT)
- Culturally Sensitive
- Dialectical (DBT)
- Eclectic
- Emotionally Focused
- Existential
- Expressive Arts
- Family / Marital
- Family Systems
- Feminist
- Forensic Psychology
- Gestalt
- Gottman Method
- Humanistic
- Hypnotherapy
- Imago
- Integrative
- Interpersonal
- Intervention
- Jungian
- Mindfulness-based (MBCT)
- Motivational Interviewing
- Multicultural
- Narrative
- Neuro-Linguistic
- Neurofeedback
- Play Therapy
- Psychoanalytic
- Psychodynamic
- Psychological Testing and Evaluation
- Relational
- Sand Play
- Solution Focused Brief (SFBT)
- Somatic
- Transpersonal
- Trauma Focused
Online Therapy
See Nearest
"I specialize in helping couples' achieve greater satisfaction in marital and committed partnerships. I am a certified Imago Relationship Therapist trained in the art of communication. I teach couples a step-by-step process to understand how to transform relationship challenges into workable solutions through improved communication."
"Theresa K. Cooke is the founder of FACES, INC. Graduating from Albion College in 1982, she went on to Hofstra University in New York to earn her Master's degree in 1984. She brings 30+ years of experience through training, certification, education and experience working with over 3000 cases."
"I have extensive clinical experience working with both chemically dependent and mental health clients. I am comfortable working with individual as well as couples in regards to various clinical challenges specific to cognitive behavioral problems. I have extensive training in dealing with anger management, clinical hypnosis, anxiety and depression, post traumatic stress disorders and counseling criminal offenders."
"HAVE YOU LOST YOUR INNER LIGHT?! If so I can help you get it back! I've compassionately guided many people of all ages and all walks of life through their struggles. Innerlight Counseling Services LLC is the culmination of years of traditional therapy techniques coupled with holistic perspectives and practices. My ultimate goal is to help my clients remember their inner strengths and achieve well-being. I strive to educate my clients on the many factors that can influence their symptoms, give them tools to manage or extinguish their symptoms, support them in the maintenance of their health and ultimately find their INNER LIGHT."
Office is near:Flint, Michigan 48502
"Hello and be encouraged as you seek a therapist who is a "good fit." Studies show that a good therapeutic relationship plays a major role in achieving successful outcomes, so time spent finding the person who is right for you is time well spent. I work with adults and teens from age 16. I am experienced with a variety of issues, including depression, anxiety, bipolar disorder, PTSD, childhood abuse (physical/emotional/sexual), borderline personality, anger management, relationship issues (couples/parent-child), grief/loss, and substance use disorders. I seek to create an environment where you feel safe, supported, and encouraged."
Office is near:Flint, Michigan 48502
No more EMDR Therapists in Flint - Try other Therapists below
"I enjoy a good challenge. I enjoy working with several different type clients. Lately I have been working with gamblers and really enjoy the challenges they present. I have a long history of working with children and know a lot about ADHD. Additionally, I enjoy discussing how GOD can change us and How to be Free. If you want to discuss your concerns please contact me. I have developed a private office where I can do a sliding scale for people who want counseling but may be limited in resources."
"The mind, body and soul are all woven together and its essential to mend what is not well in any one of these areas for the well being of the whole body. I am happy to be a part of your journey of healing yourself psychologically and emotionally. My approach to therapy differs with each individual as I believe not one technique benefits the therapeutic goals of all people."
"Are you " sick and tired of being sick and tired?" Are you tired of feeling stuck? Do you need someone to help you overcome your depression or anxiety? Do you need help on starting the next chapter of your life? Let me help you.... My goal is to help you find personal truth, healing and peace. Today is your day to re-establish your life without baggage or pain. If you're reading this, go beyond the words and give me a call today."
Flint, Michigan 48502
"Hello, I'm Heidi McMeekin, and I am a health and wellness counselor who is passionate about helping people heal the past, embrace the present, and find hope in the future! My educational background is strongly rooted in holistic health care, wellness counseling and coaching, pain management and treatment of mood disorders. My goal for all clients is the same: to help them optimize their emotional, mental, and physical health to make it possible to live their best life possible! Call today for a free face to face consultation. Flexible scheduling options. Reduced fees may be available."
"I have over 15 years of experiences working with diverse populations as an social worker. I provide individual, adolescents, family and group psychotherapy services. My specialties are working with clients who suffers from anxiety, panic attacks, and depression. I enjoy working with people and in my years practicing as a social worker, I understand the importance of providing compassion, empathy, and meeting each person where they are in life without judgment. I provide a safe environment in which allows the client to express their most inner thoughts and concerns."
"Taylor Psychological Clinic, PC is a privately held professional corporation providing comprehensive outpatient counseling services in the Flint area for over 30 years. Our clinic offers qualified counseling, providing state of the art mental health treatment to clients through psychotherapy. Our staff includes a variety of mental heatlh professionals in order to meet the specific needs of each individual. Our staff includes Psychiatrists, Psychologists, Social Workers, and Professional Counselors. Our staff has experience working with children, adolescents, and adults from a variety of cultural backgrounds. Each Health professional uses a unique Integrative approach to treatment."
Flint, Michigan 48503
"I work well with both families , relationships and individuals. I have extensive experience and training in both mental health and substance abuse issues, and work with addictive behaviors related to money, gamblinng , sexual addiction, romance addiction,. I utilize many treatment modalities and attempt to find the best individualized treatment for each client. ."
"I provide psychotherapy for depression, anxiety, women's issues, disordered eating, body image issues, self-acceptance, substance abuse/chemical dependency, relationship issues, ADHD, dementia and grief and loss. I use a holistic approach to assist my clients in overcoming barriers that get in the way of their health and well-being. My goal is to provide the help needed to live a healthy, balanced life."
"I practice primarily with individuals and couples in these distinct areas: 1) With adults and youth who have a history of or are experiencing depression and anxiety. 2) With engaged and married couples experiencing difficulties in their relationship, especially those of the Catholic/Christian faith. 3) With adults and youth who have a spouse or parent experiencing or who have experienced a substance use problem or mental illness. 4) With adults and youth who are grieving the illness or loss of a loved one."
"I provide psychotherapy for children, adolescents and adults. Some of my experience includes working with people with ADHD, abuse/neglect, divorce, anxiety, depression, relationship/family issues, disordered eating as well as transgendered clients. My therapeutic approach with children and adolescents includes play therapy, development of coping skills, and behavioral interventions in collaboration with the child's family. I use an eclectic approach to therapy when working with adults including behavioral, cognitive, and humanistic psychology."
"Some therapeutic models leave out the past, with the theory that the past isn't important. But the past does matter, as it has influenced how we became who we are. The very earliest period of our life, that from birth to three, laid the foundation for all future relationships, and for how we developed emotionally. You're seeking help because you want something to change. Uncover & name the issues, and determine your direction. Heal the past, accept responsibility for the present, and experience hope for the future."
"The great poet Rumi wrote "out beyond ideas of wrong-doing and right-doing, there is a field. I'll meet you there." Lets pretend that field is a space where calm, love and forgiveness exist. Yet, in reality what really lies in that space is a voice that gets in the way of the peace and happiness all human beings long for."
"I have worked with individuals in crisis for 25 years. I have experience in working with diverse populations and ages ranges from infants to geriatrics. My work and experiences prior have given me the knowledge and empathy that have been invaluable in my current work. I recognize that God is essential to emotional growth and healing and appreciate working from a Christian perspective and incorporating scripture into the work in accordance with client's desire. I have been a practicing clinical therapist since 2009."
""Do you feel overwhelmed and don't know who to turn to for help?" I provide individual and couples counseling in a warm and professional environment. I work with a broad spectrum of client needs including depression, anxiety, relationship/family issues, PTSD and addiction. I believe that every person is invested with strengths and abilities that make them unique. Various problems can compromise our ability to reach our full potential. You may simply need better tools in order to overcome detrimental habits or beliefs that limit you. Together we will create positive change."
"I believe that every individual is unique and that he or she will bring specific needs and goals into therapy. By using an eclectic approach to therapy, I strive to increase an individual's well-being. I work with individuals with the intention that he or she may employ coping skills in order to utilize strengths and diminish difficulties. I provide a warm and comfortable environment in which an individual is able to enhance his or her own sense of well-being."


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