Name: foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7
Uploaded: 2017-09-17T14:00:00
Description: Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Dette arbejde er støttet af K-INBRE P20GM103418, Kansas soja Kommissionen (KSC/PSU 1663), ACS PRF 56629-UNI7 og PSU polymer kemi start fond, alle SS. Vi takker Universitet Videograf, Mr. Jacob Anselmi, for hans fremragende arbejde med video. Vi takker også Mr. Roger Heckert og fru Katha Heckert for deres generøse støtte til forskning.

1. syntese og Functionalization af multi parametrisk Magneto-fluorescerende sensorer (MFnS).
<ol><li>Syntese af superparamagnetisk jernoxid nanopartikler (IONPs) <ol><li>at forberede IONP syntese, forberede de følgende 3 løsninger: løsning 1: FeCl 3 (0,70 g) og FeCl 2 i H 2 O (2 mL), løsning 2: NH 4 OH (2,0 mL, 13,4 M) i H 2 O (15 mL) og løsning 3: polyacrylic syre (0.855 g) i H 2 O (5 mL).</li>
		<li>Tilføje 90 µL 2 M saltsyre (HCl) til løsnin...

<ol>
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Denne protokol er blevet designet til at producere fuldt funktionel MFnS så enkelt som muligt. Men der er mange vigtige punkter hvor ændring af protokollen kan være nyttig, afhængigt af brugerens endelige mål. For eksempel, brug af forskellige antistoffer ville give mulighed for målretning af mange andre patogener. Desuden, er denne protokol ikke begrænset til brug af antistoffer som målretning molekyler. Ethvert molekyle, som har specifikke bindende affinitet for target patogener...

Den vedvarende forekomst af bakteriel forurening i både kommercielt fremstillede fødevarer og vandkilder har skabt et behov for mere og mere hurtig og specifik diagnostisk platforme. 1 , 2 nogle af de mere almindelige bakterielle kontaminanter ansvarlig for mad og vand kontaminering er fra Salmonella, Staphylococcus, Listeria, Vibrio, Shigella, Bacillus og Escherichia slægterne. 3 , 4 bakteriel forurening af disse patogener ofte resulterer i symptomer som feber, kolera, gastroenteritis, og diarré. 4 forurening af vandkilder ofte har drastiske og negative virkninger på Fællesskaber uden adgang til tilstrækkeligt filtreret vand, og forurening af fødevarer har ført til et stort antal sygdomme og produkt recall indsats. 5 , 6
For at mindske forekomsten af sygdomme forårsaget af bakteriel forurening, har der været en række bestræbelser på at udvikle metoder, som vand og fødevarer kan effektivt scannes før salg eller forbrug. 3 teknikker såsom PCR,1,7,8,9,10 ELISA,11,12 loop-medieret isotermisk forstærkning ( LAMPE),13,14 ,15,16,17,18,19,20,21, 22,23,24 er for nylig blevet anvendt til påvisning af forskellige patogener. Sammenlignet med traditionelle bakteriel dyrkning metoder, er disse teknikker langt mere effektive med hensyn til specificitet og tid. Men disse teknikker stadig kamp med falske positiver og negativer, komplekse procedurer og omkostninger. 1 , 3 , 25 det er netop derfor at multiparametric magneto-fluorescerende sensorer (MFnS) er foreslået som en alternativ metode til registrering af bakteriel.
Disse sensorer par unikt sammen magnetiske afslapning og fluorescerende modaliteter, giver mulighed for en dobbelt-registrering platform, der er både hurtig og præcis. Ved hjælp af E. coli O157: H7 som prøve forurenende stof, er MFnS evne til at afsløre så lidt som 1 CFU inden for minutter påvist. Patogen-specifikke antistoffer bruges til at øge specificitet, og kombinationen af både magnetiske og fluorescerende modaliteter giver mulighed for påvisning og kvantificering af bakterielle kontaminanter i både lav - og høj-kontaminering intervaller. 16 for bakteriel forurening, vil sensorer sværmer omkring bakterier på grund af de målretning evner af patogen-specifikke antistoffer. Bindingen mellem den magnetiske sensorer og bakterier begrænser samspillet mellem den magnetiske jernkerne og de omkringliggende vand protoner. Dette medfører en stigning i T2 afslapning gange, som er registreret af en magnetisk relaxometer. Som koncentrationen af bakterier i løsning stiger, sprede sensorer med det øgede antal bakterier, resulterer i lavere T2 værdier. Omvendt, fluorescens emission vil stige i andelen med koncentrationen af bakterier på grund af det øgede antal sensorer direkte bundet til patogen. Centrifugering af prøverne, og isolering af den bakterielle pellet, vil kun spare nanopartikler direkte knyttet til bakterier, at fjerne enhver frit svævende sensorer, og direkte korrelerede fluorescens emission med antallet af bakterier er til stede i løsning. En skematisk gengivelse af denne mekanisme er repræsenteret i figur 1.
Denne MFnS platform er designet med punkt af pleje screening i tankerne, resulterer i lave omkostninger og bærbare egenskaber. MFnS er stabilt ved stuetemperatur, og er kun nødvendig i meget lave koncentrationer for nøjagtig påvisning af bakterielle kontaminanter. Desuden, efter syntese, brug af MFnS er enkel og kræver ikke brug af uddannede fagfolk på området. Endelig, denne diagnostiske platform giver mulighed for meget tilpasselig målretning giver dig mulighed for ved denne én platform kan bruges til at registrere patogener i alle former, i mange forskellige indstillinger.

<table border="0" cellpadding="0" cellspacing="0" width="821">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferrous Chloride Tetrahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">I90-500</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferric Chloride Hexahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>I88-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ammonium Hydroxide</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A669S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Hydrochloric Acid</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A144S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Polyacryllic Acid</td>
			<td style="width:156px;">Sigma-Aldrich</td>
			<td style="width:197px;">323667-100G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">EDC</td>
			<td style="width:156px;">Thermofisher Scientific</td>
			<td style="width:197px;">22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">NHS</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>AC157270250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O111 antibody&#160;</td>
			<td style="width:156px;">sera care</td>
			<td>5310-0352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O157:H7 antibody [P3C6]&#160;</td>
			<td style="width:156px;">Abcam</td>
			<td style="width:197px;">ab75244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">DiI Stain</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">D282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nutrient Broth</td>
			<td style="width:156px;">Difco</td>
			<td style="width:197px;">233000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-dried E. coli O157:H7 pellet</td>
			<td style="width:156px;">ATCC</td>
			<td style="width:197px;">700728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Relaxomteter&#160;</td>
			<td style="width:156px;">Bruker</td>
			<td style="width:197px;">mq20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Zetasizer</td>
			<td style="width:156px;">Malvern</td>
			<td style="width:197px;">NANO-ZS90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plate Reader&#160;</td>
			<td style="width:156px;">Tecan</td>
			<td style="width:197px;">Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Column&#160;</td>
			<td style="width:156px;">QuadroMACS</td>
			<td style="width:197px;">130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge</td>
			<td style="width:156px;">Eppendorf</td>
			<td style="width:197px;">5804 Series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge (accuSpin Micro 17)</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">13-100-676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Floor Model Shaking Incubator</td>
			<td style="width:156px;">SHEL LAB</td>
			<td style="width:197px;">SSI5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Analytical Balance</td>
			<td style="width:156px;">Metler Toledo</td>
			<td style="width:197px;">ME104E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Digital Vortex Mixer</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-215-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Open-Air Rocking Shaker</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-217-765</td>
			<td></td>
		</tr>
	</tbody>
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foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

Enterohemorrhagic Escherichia coli O157: H7 er blevet forbundet med både vandbårne og fødevarebårne sygdomme, og er stadig en trussel på trods af mad - og vand-screeningsmetoder bruges i øjeblikket. Mens konventionelle bakteriel påvisningsmetoder, såsom Polymerasekædereaktionen (PCR) og enzym-forbundet immunosorbent assays (ELISA) specifikt registrerer patogene stoffer, kræver de omfattende prøveforberedelsen og lange ventetider. Derudover disse praksis kræver avancerede laboratorieinstrumenter og indstillinger, og skal udføres af uddannede fagfolk. Heri, foreslås en protokol for en enklere diagnostisk teknik, der er udstyret med den unikke kombination af magnetiske og fluorescerende parametre i en nanopartikel-baseret platform. Den foreslåede multiparametric magneto-fluorescerende sensorer (MFnS) kan finde E. coli O157: H7 kontaminering med så lidt som 1 kolonidannende enhed i løsning inden for mindre end 1 time. Derudover MFnS evne til at forblive meget funktionelle i komplekse medier såsom mælk og søvand er blevet verificeret. Yderligere specificitet assays blev også brugt til at demonstrere evne til MFnS at kun opdage de specifikke målbakterierne, selv i nærværelse af lignende bakteriearter. Parring af magnetiske og fluorescerende modaliteter giver mulighed for påvisning og kvantificering af patogenet forurening i en bred vifte af koncentrationer, udstiller sine højtydende i både tidlige - og sene forurening påvisning. Effektivitet, overkommelighed og overførsel af MFnS gøre dem en ideel kandidat til punkt af pleje screening for bakterielle kontaminanter i en bred vifte af indstillinger, fra akvatiske reservoirer til kommercielt emballerede fødevarer.

MFnS virkningsmekanisme er repræsenteret i figur 1. Klynger af MFnS omkring overfladen af bakterielle kontaminanter forstyrrer interaktioner mellem magnetic cores af MFnS og de omkringliggende brint kerner. Som følge af denne klyngedannelse, magnetiske afslapning øge værdier. Da koncentrationen af bakterielle kontaminanter stiger, klyngedannelse reducerer, og ændringen i T2 værdier falder. Tilføjelse af en fluorescerende modalitet er derfor afgørende. Som bakteriel koncentrationen st...

Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Det overordnede mål med denne protokol er at syntetisere funktionelle sensorer på bærbar, omkostningseffektive, og hurtig påvisning af specifikt målrettet sygdomsfremkaldende bakterier gennem en kombination af magnetiske afslapning og fluorescens emission modaliteter.

Fødevarebårne patogen Screening ved hjælp af Magneto-fluorescerende Nanosensor: Hurtig påvisning af E. Coli O157: H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Research

JoVE Journal

Immunology and Infection

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

En-dags Workflow ordningen for bakteriepatogen Detection og antimikrobiel resistens fra blodkulturer

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The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

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Hurtig screening af HIV revers transkriptase og Integrase hæmmere

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

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Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

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Hurtig Molekylær Detection og Differentiering af Influenza Virus A og B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

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Oprettelsen af en tætte Transposon indsættelse bibliotek ved hjælp af bakteriel konjugation i Enterobacterial stammer som Escherichia Coli eller Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

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Hurtig, sikker og simpel manuel sengelamper nukleinsyre udvinding til påvisning af Virus i blodprøver

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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En høj overførselshastighed Platform for Screening af Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...