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PARTE I: IDENTIFICAZIONE PATHOGEN 1. Preparazione del campione Nota: L&#39;intero flusso di lavoro molecolare, come è descritto nel seguente protocollo deve essere eseguita secondo le raccomandazioni per la garanzia della qualità nella diagnostica molecolare 3. <ol><li> Aggiungere una aliquota 0,1 ml di coltura di sangue a 0,9 ml di NaCl allo 0,9% in una provetta da 1,5 ml di reazione per diventare un campione diluito 1:10. (Diluire al fine di impedire l&#39;inibizione qPCR). </li><li> Centrifugare il campione a 13.400 xg per 5 minuti a pellet del DNA batterico. </li><li> Risospendere il pellet batterico in 100 microlitri sterile demineralizzata H 2 O. </li><li> Conservare il campione di DNA a 4 ° C fino ad ulteriore utilizzo. </li></ol> 2. Identificazione Assay: Real-time PCR 16S rDNA <ol><li> Preparare le miscele di reazione come segue. Il test consiste di quattro reazioni separate per campione. Ogni miscela contiene 12,50 maestro plmix, 0,9 uM avanti di primer (5-TCCTACGGGAGGCAGCAGT-3) 7, primer 0,6 micron inverso (5-GGACTACCAGGGTATCTAATCCTGTT-3) 8 e un pannello di sonde. La quantità di sonde è dato per ciascuno dei quattro reazioni separate in seguito. </li><li> La prima reazione comprende: <ul><li> 0,2 pM sonda universale (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) 8 </li><li> 0,2 pM P. aeruginosa sonda (5-JOE-CCAAAACTACTGAGCTAGAGTACG-3-BHQ1) </li></ul></li><li> La seconda reazione comprende: <ul><li> 0,2 pM E. coli sonda (5-JOE-GGAGTAAAGTTAATACCTTTGCTCATT-3-BHQ1) </li><li> 0,2 pM Pseudomonas spp. Sonda (5-NED-CCTTCCTCCCAACTTAAAGTGCTT-3-MGBNFQ) </li></ul></li><li> La terza reazione comprende: <ul><li> 0,2 pM Staphylococcus spp. Sonda (5-NED-AATCTTCCGCAATGGGCGAAAGC-3-MGBNFQ) </li><li> 0,2 pM S. aureus sonda (5-FAM-AGATGTGCACAGTTACTTACACATAT-3-BHQ1) </li><li> 0,2 uM Enterococcus spp. (5-JOE-TCCTTGTTCTTCTCTAACAACAGAG-3-BHQ1) </li></ul></li><li> La quarta reazione comprende: <ul><li> 0,2 pM sonda universale (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) </li><li> 0,3 pM Streptococcus spp. Sonda (5-NED-CCAGAAAGGGACSGCTAACT-3-MGBNFQ) </li><li> 0,2 pM S. pneumoniae sonda (5-JOE-CCAAAGCCTACTATGGTTAAGCCA-3-BHQ1) </li></ul></li><li> Aggiungere demineralizzata sterile H 2 O a raggiungere un volume totale di 20 pl. Aggiungere 20 pl di ciascuna miscela di reazione ai pozzetti di una piastra a 96 pozzetti PCR. </li><li> Aggiungere 5 pl di campione in ciascun pozzetto. </li><li> Usare un film adesivo per sigillare la piastra a 96 pozzetti PCR. </li><li> Eseguire la piastra sul ABI PRISM 7900HT sistema in tempo reale PCR utilizzando le seguenti condizioni ottimali cicli termici: <ul><li> Pre-riscaldamento a 50 ° C per 10 min </li><li> Denaturazione iniziale a 95 ° C per 15 min </li><li> 42 cicli di <ul type="circle"><li> Denaturazione a 95 ° C per 15 s </li><li> Ricottura a 60 ° C per 1 min </li></ul></li></ul></li></ol> 3. Analisi dei risultati <ol><li> Regolare la soglia del Analysis Ct a 0,1 nelle impostazioni di analisi della scheda. Limitare le configurazioni di base per iniziare (ciclo): 6 e End (Cycle): 15. </li><li> Registrare il ciclo soglia (Ct) il valore per tutti i campioni. Il valore di cut-off di considerare un risultato positivo come PCR può essere impostato ad un valore di Ct-35. La quantità di batteri presenti in colture di sangue variava da 10 luglio - 10 Novembre CFU / ml, generando Ct valori inferiore a 35. </li></ol> PARTE II: PROVE DI SENSIBILITA &#39;AGLI ANTIBIOTICI 4. Isolamento di batteri da emocolture positive 9 <ol><li> Aspirare 5 ml di brodo da una bottiglia coltura positiva sangue e trasferirlo in un tubo separatore siero. </li><li> Centrifugare la provetta siero separatore a 2000 xg per 10 min. </li><li> Eliminare il surnatante dal tubo separatore siero. </li><li> Transfer bacbatteri dallo strato di gel del tubo con un tampone di cotone sterile in soluzione fisiologica 0,9% fino a McFarland 0,5 sospensione titolata è ottenuta. </li></ol> 5. Inoculazione di Micro Piastre Titre <ol><li> Diluire il 0,5 sospensione McFarland in doppia concentrato Mueller Hinton II brodo per formare una sospensione di 5 x 10 5 CFU / ml. </li><li> Aggiungere questa sospensione ai pozzetti di una piastra di micro titolo contenente una selezione di antibiotici (Tabella 1). </li><li> Incubare la piastra micro titolo a 37 ° C per 6 h. </li><li> Conservare una aliquota della sospensione a 4 ° C (come controllo negativo della crescita). </li><li> Dopo 6 ore di incubazione, trasferire il contenuto di ciascun pozzetto in una provetta sterile, così come il campione negativo di controllo della crescita che è stato conservato a 4 ° C. </li><li> Centrifugare le provette a 16000 xg per 5 min. </li><li> Rimuovere con cautela il surnatante, senza disturbare il pellet batterico. </li><li> Risospendere il pellet in H sterile demineralizzata <sub&gt; 2 O. </li><li> Diluire i campioni di 10 volte in sterile demineralizzata H 2 O. </li></ol> 6. Real-time PCR 16S rDNA 10 <ol><li> Preparare la miscela di PCR come segue: <ul><li> 12,50 ul iQ SYBR Green Supermix </li><li> 0,5 pM innesco in avanti 16S-1 (5-TGGAGAGTTTGATCCTGGCTCAG-3) 11 </li><li> 0,25 16S-2 pM primer reverse (5-TACCGCGGCTGCTGGCAC-3) 11 </li><li> demineralizzata sterile H 2 O a un volume totale di 20 pl </li></ul></li><li> Aggiungere 20 pl di miscela PCR ai pozzetti di una piastra a 96 pozzetti PCR. </li><li> Aggiungere 5 pl di campione in ciascun pozzetto. </li><li> Usare un film adesivo per sigillare la piastra a 96 pozzetti PCR. </li><li> Esegui il piatto sul singolo colore MyiQ Real-Time PCR Detection System, usando le seguenti condizioni ottimali cicli termici: <ul><li> Denaturazione iniziale a 95 ° C per 4 min </li><li> Ricottura iniziale a 65 ° C per 30 s </li><li> 35 cicli di <ul><li> Denaturazione a 95 ° C per 15 s </li><li> Ricottura a 60 ° C per 1 min </li></ul></li><li> Melt analisi della curva (60-95 ° C in 20 min con incrementi di 0,57 ° C) </li></ul></li></ol> 7. Analisi dei risultati <ol><li> Calcolare il cut-off Ct utilizzando una delle seguenti formule (a seconda del tipo di antibiotico). <ul><li> In generale: Cut-off Ct = valore Ct valore positivo controllo della crescita + 0,5 x (valore negativo Ct controllo della crescita - il valore Ct positivo controllo della crescita) </li><li> Piperacillina, piperacillina / tazobactam e ceftazidime in gram-negativi, Amoxicillina, oxacillina e trimetoprim / sulfametossazolo in S.aureus, amoxicillina in Enterococcus spp. Cut-off Ct = valore Ct positivo controllo della crescita + 0,25 x (Ct crescita del valore negativo controllo - il valore Ct positivo controllo della crescita) </li></ul></li><li> Utilizzare il campione incubato con sterile demineralizzata H 2 O, come controllo della crescita positivo.</li><li> A seconda del microrganismo, utilizzare il controllo appropriato crescita negativa: <ul><li> Gram-negativi Esempio aste incubate con miscela di antibiotici </li><li> Enterococcus spp. Campione conservato a 4 ° C </li><li> Esempio S.aureus conservati a 4 ° C </li></ul></li><li> Determinare la suscettibilità (S) o resistenza (R) del ceppo per l&#39;antibiotico testate come segue: <ul><li> Un valore Ct superiore al valore di cut-off Ct indica suscettibilità </li><li> Un valore Ct inferiore al valore di cut-off Ct indica la resistenza </li></ul></li></ol> 8. Risultati rappresentativi Due organismi modello, cioè un Gram-negativo E. coli e Gram-positivi S. aureus, vengono scelti per visualizzare la procedura combinata per il rilevamento e l&#39;identificazione di patogeni batterici e la determinazione del loro profilo antimicrobica. La prima parte del protocollo comprende l&#39;identificazione patogeno. Spesonde specifici sono progettate per il rilevamento di otto microrganismi clinicamente rilevanti. In presenza di un bersaglio incluso nel pannello batterica, curve di amplificazione vengono generati e valori di Ct sono calcolati (Figura 2). Il valore di cut-off di considerare un risultato positivo PCR come è impostata su un valore di Ct 35. In figura 2A, il profilo di identificazione di un E.coli cultura infettata ematico. La sonda 16S universale è incluso in due miscele di reazione separati e genera di conseguenza due curve di amplificazione (Ct di 25,20 e 25,95). Il terzo segnale è derivato dalla sonda specifica per E. coli (Ct di 27,04). L&#39;identificazione di un S. aureus-cultura sangue infetto è mostrato nella figura 2B. La sonda universale 16S ha acquisito il segnale di amplificazione di 33,35 e 33,71. I due segnali rimanenti sono derivate dalle sonde specifiche per Staphylococcus spp. e S. aureus (Ct di 32,48 e 30,59). Figura 3 è un esempio di un antibiotico trama amplificazione test di sensibilità, che rappresenta il ceppo E. coli che è stato anche dimostrato nella Figura 2A. Ogni riga rappresenta un antibiotico che il campione è stato incubato con batterica. Un campione con un valore basso Ct è un esempio in cui la crescita si è verificata in presenza di un antibiotico, indicando resistenza all&#39;antibiotico testato. Al contrario, un valore elevato Ct rappresenta un campione in cui si è verificata alcuna crescita a causa del funzionamento efficace del antibiotico, indicando suscettibilità all&#39;antibiotico testato. La Tabella 1 illustra la determinazione del profilo antimicrobica della E. coli e S. aureus isolati. Tutti i valori sono riportati Ct e, utilizzando le formule indicate nel testo del protocollo (7.1), due cut-off valori Ct sono calcolatiregolamentato di distinguere tra resistenza e suscettibilità. Il ceppo è resistente all&#39;antibiotico se il valore riportato Ct è inferiore al calcolato cut-off Ct (e viceversa). <img alt="Figura 1" src="/files/ftp_upload/3254/3254fig1.jpg" /> Figura 1. Diagramma di flusso del patogeno identificazione e test di suscettibilità agli antibiotici procedure utilizzando real-time PCR 16S rDNA. <img alt="Figura 2" src="/files/ftp_upload/3254/3254fig2.jpg" /> Figura 2 Identificazione dosaggio:. Trame di amplificazione e valori ciclo soglia (valori di Ct) Una coltura di sangue positiva viene rilevata dal universale 16S rDNA sonda, mentre le sonde specifiche vengono utilizzati per l&#39;identificazione del patogeno causale.. Amplificazione plot A. della cultura sangue contenente E. coli; amplificazione trama B. della cultura sangue contenente S. aureus, pseudo ae, Pseudomoncome aeruginosa; uni, 16S sonda universale; Ecoli, Escherichia coli sonda; pseudo sp, Pseudomonas spp. sonda; S. pneu, Streptococcus pneumoniae sonda; Strep sp, Streptococcus spp. sonda; Entero, Enterococcus spp. sonda; S. aureus, Staphylococcus aureus sonda; Staph sp, Staphylococcus spp. sonda. <img alt="Figura 3" src="/files/ftp_upload/3254/3254fig3.jpg" /> Figura 3. Plot di amplificazione delle prove di suscettibilità agli antibiotici di E. coli isolare (campione 1). Ciascuna curva rappresenta un antibiotico che il ceppo è stato incubato con. Un primo segnale è causata da un elevato carico batterico, il che significa che il ceppo è cresciuto in presenza dell&#39;antibiotico testato ed è quindi resistente all&#39;antibiotico. Segnali tardiva indicano che il ceppo non è cresciuto in presenza dell&#39;antibiotico, in altre parole, esso è suscettibile. <table border="1"><tbody&gt; <tr><td> Esempio 1: E. coli </td><td></td><td></td><td> Esempio 2: S. aureus </td><td></td><td></td></tr><tr><td> AST </td><td> Ct </td><td> R / S </td><td> AST </td><td> Ct </td><td> R / S </td></tr><tr><td> Amoxicillina 8 mg / L </td><td> 16,83 </td><td> R </td><td> Amoxicillina 0,25 mg / L </td><td> 21,03 </td><td> R </td></tr><tr><td> Amoxicillina-acido clavulanico 8/4 mg / L </td><td> 17,36 </td><td> R </td><td> Oxacillin 2 mg / L </td><td> 25,80 </td><td> S </td></tr><tr><td> Piperacillina 16 mg / L </td><td> 16,67 </td><td> R </td><td> Vancomicina 2 mg / L </td><td> 25,20 </td><td> S </td></tr><tr><td> Piperacillina-tazobactam 16 /4 mg / L </td><td> 24,15 </td><td> S </td><td> Gentamicina 4 mg / L </td><td> 25,86 </td><td> S </td></tr><tr><td> Ciprofloxacina 1 mg / L </td><td> 29,72 </td><td> S </td><td> Trimetoprim-sulfametossazolo 2/38 mg / L </td><td> 24,62 </td><td> S </td></tr><tr><td> Ceftazidime 1 mg / L </td><td> 24,03 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Ceftazidime 8 mg / L </td><td> 26,58 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Gentamicina 4 mg / L </td><td> 29,83 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Trimetoprim-sulfametossazolo 2/38 mg / L </td><td> 27,60 </td><td> S </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Controllo della crescita negativo (miscela di antibiotici) </td><td> 30,41 </td><td></td><td> Controllo della crescita negativo (campione conservato a 4 ° C) </td><td> 27,42 </td><td></td></tr><tr><td> Controllo positivo di crescita </td><td> 16,90 </td><td></td><td> Controllo positivo di crescita </td><td> 20,22 </td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Cut-off Ct-valore 1 * </td><td> 21,76 </td><td></td><td> Cut-off Ct-valore 1 *** </td><td> 23,82 </td><td></td></tr><tr><td> Cut-off Ct-valore 2 ** </td><td> 18,75 </td><td></td><td> Cut-off Ct-valore 2 **** </td><td> 22,02 </td><td></td></tr><tr><td> * Per amoxicillina, amoxicillina-acido clavulanico, ciprofloxacina, gentamicin, trimetoprim-sulfametossazolo ** Per piperacillina, piperacillina-tazobactam, ceftazidime </td><td></td><td></td><td> *** Per vancomicina e gentamicina **** Per amoxicillina, oxacillina e trimetoprim-sulfametossazolo </td><td></td><td></td></tr></tbody></table> Tabella 1. Determinazione del test di sensibilità agli antibiotici dei due campioni (E. coli e S. aureus). Valori Ct della PCR-analisi sono stati copiati a questo file excel, in quanto in grado di calcolare automaticamente i due cut-off ALORI Ct dal controllo di crescita positivo e negativo, utilizzando le formule indicate nel testo del protocollo. Se un antibiotico mostra un valore Ct inferiore al valore soglia Ct, il ceppo è resistente all&#39;antibiotico, se il valore Ct è maggiore del cut-off, il ceppo è sensibile.

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Il protocollo qui descritto permette la rapida identificazione di agenti patogeni e fornisce un profilo funzionale antimicrobico che potrebbe portare alla somministrazione precoce di antibiotici adeguati, migliorando così la prognosi dei pazienti con infezioni del sangue. A seconda delle condizioni richieste di test, cioè a basso costo, velocità elevata, minimo tempo di ritorno, condizioni di prova può essere regolata. L&#39;intera procedura può essere eseguita entro un giorno lavorativo. Inoltre, le due parti del protocollo possono essere eseguite contemporaneamente, che riduce il tempo di ritorno in modo significativo. Come qui presentata, il pannello di identificazione è una selezione dei batteri più clinicamente rilevanti nel nostro ospedale. Poiché il principio fondamentale è il targeting del gene 16S regione, sonde specifiche per altri microrganismi possono essere progettati e aggiunte al saggio. Il dosaggio completo era originariamente prevista per l&#39;analisi rapida di emocolture, ma può anche essere utilizzato per la trasformazione di oltre campioni di materiali. Questo è anche il caso per gli antibiotici che sono stati utilizzati per i test di sensibilità agli antibiotici: antibiotici più o di altro tipo possono essere aggiunti, in base a pattern di resistenza locali e le linee guida.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Cloruro di sodio (NaCl) </td><td> Merck Chemicals </td><td> 106404 </td><td> 0,9% in acqua </td></tr><tr><td> Vacutainer SST Siero Tubo Separator 5 ml </td><td> BD Diagnostic Systems </td><td> 367986 </td><td></td></tr><tr><td> Mueller Hinton II brodo </td><td> BD Diagnostic Dystems </td><td> 212322 </td><td> 44 g / L in acqua </td></tr><tr><td> Centrifugare Rotixa 50 rs </td><td> Andreas Hettich GmbH &amp; Co. KG </td><td> 4910 </td><td></td></tr><tr><td> Centrifuga 5415 D </td><td> Eppendorf </td><td> Fuori </td><td></td></tr><tr><td> Primers </td> &lt;td&gt; Sigma-Aldrich </td><td> na </td><td></td></tr><tr><td> Sonde </td><td> Sigma-Aldrich/Applied Biosystems </td><td> na </td><td></td></tr><tr><td> TaqManEnvironmental master mix 2,0 </td><td> Applied Biosystems </td><td> 4396838 </td><td></td></tr><tr><td> iQ SYBRGreen Supermix </td><td> Bio-Rad Laboratories BV </td><td> 170-8880 </td><td></td></tr><tr><td> MicroAmp ottica piastra a 96 pozzetti di reazione </td><td> Applied Biosystems </td><td> N8010560 </td><td></td></tr><tr><td> MicroAmp Film adesivo ottico </td><td> Applied Biosystems </td><td> 4311971 </td><td></td></tr><tr><td> iQ 96 pozzetti PCR Piastre </td><td> Bio-Rad Laboratories BV </td><td> 223-9441 </td><td></td></tr><tr><td> Microseal B Adesivo Seals </td><td> Bio-Rad Laboratories BV </td><td> MSB-1001 </td><td></td></tr><tr><td> Real-time PCR Detection System </td><td> Applied Biosystems </td><td> ABI PRISM 7900HT </td><td></td></tr><tr><td> Real-Time PCR Detection System </td><td> Bio-Rad Laboratories BV </td><td> MyiQ singolo-Color </td><td></td></tr></tbody></table>

one-day workflow scheme for bacterial pathogen detection and antimicrobial resistance testing from blood cultures

Infezioni del sangue sono associati con alti tassi di mortalità a causa della manifestazione probabile di sepsi, sepsi grave e shock settico 1. Pertanto, la somministrazione rapida di una terapia antibiotica è di primaria importanza nel trattamento delle infezioni del sangue. L&#39;elemento fondamentale in questo processo è di temporizzazione, fortemente dipendente dai risultati di identificazione batterica e test di sensibilità agli antibiotici. Entrambi questi parametri sono comunemente ottenuti dalla cultura test basato, che richiede molto tempo e richiede in media 24-48 ore 2, 4. Lo scopo dello studio era di sviluppare saggi a base di DNA di identificazione rapida di infezioni del sangue, nonché un rapido test di suscettibilità antimicrobica. Il primo saggio è una 16S rDNA eubacterial-based real-time PCR integrate con specie o dei generi di sonde specifiche 5. Utilizzando queste sonde, i batteri Gram-negativi compreso Pseudomonas spp., Pseudomonas aeruginosae Escherichia coli, nonché batteri gram-positivi compresi Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., e Streptococcus pneumoniae potrebbero essere distinti. Usando questo saggio multisonda, una identificazione prima della causativo microrganismo è stato dato dopo 2 ore. In secondo luogo, abbiamo sviluppato un semi-test molecolare per i test di sensibilità agli antibiotici di S. aureus, Enterococcus spp. e (facoltativo) aerobi gram-negativi 6. Questo saggio è stato basato su uno studio in cui venne usato PCR per misurare la crescita di batteri 7. I batteri raccolti direttamente da colture di sangue vengono incubate per 6 h con una selezione di antibiotici, e dopo un SYBR Green basato real-time PCR determina inibizione della crescita. La combinazione di questi due metodi possono indirizzare la scelta di una terapia antibiotica idonea lo stesso giorno (Figura 1). Insomma, L&#39;analisi molecolare sia di identificazione e sensibilità agli antibiotici offre una alternativa più veloce per il rilevamento degli agenti patogeni e potrebbe migliorare la diagnosi delle infezioni del sangue.

Watch this Scientific Journal Video about One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures at JoVE.com

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

La progettazione di un semplice schema di un giorno di workflow per la diagnostica dei patogeni batterici permette il rapido riconoscimento di infezioni del sangue. L&#39;inclusione di otto obiettivi clinicamente rilevanti batteriche ed i loro profili di resistenza agli antibiotici offre al medico una prima idea il giorno stesso, che può portare ad una terapia più adeguata.

Un giorno Scheme flusso di lavoro per batterica Rilevazione dei patogeni e test di resistenza antimicrobica dalle emocolture

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

one-day-workflow-scheme-for-bacterial-pathogen-detection

Research

JoVE Journal

Immunology and Infection

25.2K Views.  Maastricht University Medical Center. La progettazione di un semplice schema di un giorno di workflow per la diagnostica dei patogeni batterici permette il rapido riconoscimento di infezioni del sangue. L'inclusione di otto obiettivi clinicamente rilevanti batteriche ed i loro profili di resistenza agli antibiotici offre al medico una prima idea il giorno stesso, che può portare ad una terapia più adeguata.

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

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Protocollo per ricombinante RBD-based Vaccines SARS: Preparazione di proteine, vaccinazione degli animali e la rivelazione di neutralizzazione

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

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Immunologia e infezioni

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Una procedura 1,5 ore per l&#39;identificazione di Enterococcus Specie Direttamente da emocolture

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Invasione di cellule umane da un agente patogeno batterico

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

High-throughput Metodo di rilevazione per il virus dell&#39;influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

Nanomeccanica di interazioni farmaco-bersaglio e di rilevamento di resistenza antibatterica

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR per la tipizzazione di stafilococco cassette cromosomiche Mec Tipi I a V, resistente alla meticillina<em&gt; Staphylococcus aureus</em

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Preparazione di un Sangue cultura pellet per il Rapid batterica identificazione e test di sensibilità agli antibiotici

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isotermico di amplificazione (lampada) Assays Loop-mediata per rilevamento delle specie-specifica<em&gt; Eimeria</em&gt; Che infettano Polli

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