The funding for this research project was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) via a Discovery Grant to YL.

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The authors have nothing to disclose.

התרגום של הפעולה של פעילות מחשוף RNA של DNAzyme חיידק-מגיב מבחן לקמוס מתאפשר באמצעות השימוש באוראז ופרדה מגנטית, כפי שמודגם על ידי איור 1. למרות ההפגנה של נייר לקמוס השונה לגילוי חיידקים היא נעשה עם E. coli תלוי RNA-ביקוע DNAzyme, 5,19,20 העיצוב יכול להתארך באופן כללי לכל DNAzyme ביקוע-RNA. בהינתן הזמינות הרבה של DNAzymes ביקוע-RNA עבור analytes שונים ומתודולוגיות שונות לבודד DNAzymes חדש ביקוע-RNA מבריכות אקראית רצף עבור מטרות חדשות, אנו מצפים כי פלטפורמת מבחן לקמוס שונה יכול להתארך עד זיהוי של מטרות מגוונות של עניין . מבחן לקמוס עבור E. זיהוי קולי יכול לזהות תאים 5,000 ל 500 כאשר זמן התגובה הדיווח מוגדר להיות hr 1 ו -2, בהתאמה. תגובת השרשרת של פולימראז פופולרי (PCR)כריך אנזים צמוד assay immunosorbent (ELISA) שיטות יכולות להשיג לתחום גילוי של כ 10 4 -10 5 א קולי תאים בזמני בדיקות דומים. 25,26 לפיכך, המבחן לקמוס חיידקים מציע רגישויות גילוי דומות. למרות הבדיקה לקמוס חיידקים קלה לבצע והוא יכול לייצר שינויי צבעי חיים, מספר גורמים יכולים להשפיע על תוצאות בדיקה באופן משמעותי. ראשית, האיכות באוראז חשובה מאוד. השתמשנו באוראז ממקורות שונים ומצאתי את תוצאות הבדיקה יכולה להשתנות באופן משמעותי. אנו ממליצים על השימוש באוראז מהמקור מפורט בסעיף החומרים. ההרכבה של DNAzyme / באוראז / חרוזים מגנטיים צריכה תשומת לב מיוחדת. שטיפה יסודית של חרוזים מגנטיים להסיר UrDNA unhybridized נחוצה כדי למנוע תוצאות חיוביות שגויות. טיפול גם צריך לקחת כדי למנוע הצטברות של חרוזים מגנטיים שיורית על פני השטח הפנימי של lid של הצינור microfuge, אשר עשוי להיות קשה לראות. ברגע שיש, החרוזים המגנטיים לא חשופים יותר זמן הפרדה מגנטית וכך, יכולים לשאת כמה UrDNA unhybridized שיכולים להוביל אותות חיוביים שגויים תגובת הכתב. כמו כן, חשוב להימנע מיציאת צינור microfuge על המדף המגנטי למשך זמן ארוך יותר מ -10 דקות במהלך שלב הפרדה המגנטי. החרוזים יכולים לצבור או מקל על צינור microfuge, אשר עשוי להפחית את יעילות הכביסה להציג תצווה ל-תצווה עקביות. הכללה של 0.01% Tween-20 בפתרון הכביסה יכולה לשפר את האחידות תצווה ל-תצווה והיא צריכה להיות מיושמת. חרוזים מגנטיים הם מצופים streptavidin, אשר שימש כעוגן להרכיב conjugates DNAzyme-באוראז על חרוזים מגנטיים. שניהם streptavidin ו באוראז הם מולקולות חלבון שניתן מפוגל במהלך האחסון. אנחנו בדרך כלל לאחסן את החרוזים DNAzyme-באוראז-מגנטי התאספו בבית 4 ° C עד 4 שבועותולעשות קבוצות טריות באופן קבוע כדי להשיג תוצאות עקביות יותר. טיפול גם צריך לנקוט כדי למנוע לקיחת חרוזים מגנטיים בטעות בשלב הפרדה מגנטי (שלב 6.9) לאחר הפעלת DNAzyme. מניסיוננו, פסולת הסלולר וחלקיקים אחרים הפתרון יכולים להפחית את יעילות הפרדה המגנטית, ולכן, כמה חרוזים מגנטיים ניתן בכוונה להוציא במהלך pipetting. זה יצמצם את תוצאות חיוביות שגויות. אנו ממליצים על הצעדים הבאים כדי להקל על הבעיה: זמן הפרדה יותר (כגון 5-10 דקות), שחרור איטי של לחץ על פיפטה לאפשר נסיגה עדינה של supernatant, והכפיף את supernatant סבב נוסף של פרדה מגנטית . לבסוף, חשוב כדי למנוע זיהום תאונה של פתרון כתב המניות על ידי באוראז במהלך ניסוי שבו דגימות מרובות נבדקות. בהתחשב תגובתיות גבוהה של urease, זיהום מסוג זה יכול להוביל לתוצאות חיוביות שגויות.

Bacterial pathogens are one of the major causes of global morbidity and mortality. Outbreaks from hospital-acquired infections, food-borne pathogens, and bacterial contaminants in the environment pose serious and on-going threats to public health and safety. To prevent these outbreaks, effective tools are needed that permit pathogen detection in a timely fashion under a variety of settings. Simple but still effective tests that are portable and cost-effective are greatly coveted, especially in regions that are susceptible to outbreaks but cannot afford expensive testing facilities.1-3 Although there exists a multitude of methods to detect bacteria, many of them are not suitable as screening or on-site testing tools because they require long test times, expensive instruments and complicated testing procedures. 
Colorimetric tests are particularly attractive for point-of-care or field applications as color changes can be easily detected by the naked eye. The litmus test for pH is simple, quick, and effective. Although it is a very old technology, it is still widely used today because of its simplicity and effectiveness. Surprisingly, this simple test had never been modified to achieve the detection of other analytes before we recently developed an approach of modifying this test for E. coli testing.4
The expanded litmus test for E. coli employs three additional components: an E. coli activated RNA-cleaving DNAzyme (EC1), 5 urease, and magnetic beads. DNAzymes refer to synthetic single-stranded DNA molecules with catalytic activity.6 They can be isolated from random-sequence DNA pools using in vitro selection.7,8 They are highly stable and can be produced cost-effectively using high-efficiency automated DNA synthesis.9 For these reasons, DNAzymes, particularly RNA-cleaving DNAzymes, have been widely examined for biosensing applications.6,10,11 RNA-cleaving DNAzyme sensors have been developed to detect metal ions,12-16 small molecules,17,18 bacterial pathogens5,19-21 and cancer cells.22 Given the great availability of target-induced RNA-cleaving DNAzymes, any assay that utilizes a DNAzyme can be potentially expanded to detect a diverse range of analytes.
Urease is chosen for its ability to hydrolyze urea into ammonia,23,24 resulting in a pH increase. Urease is also highly efficient, stable and amenable for conjugation to other biomolecules. Therefore, we postulated that a conjugate of an RNA-cleavage DNAzyme with urease would allow the use of litmus test for the detection of other targets.5
The action of the RNA-cleaving DNAzyme is relayed to urease-mediated increase of pH through the use of magnetic beads that are immobilized with the DNAzyme-urease conjugate. Because the activity of the DNAzyme under investigation is strictly dependent on E. coli, the presence of this bacterium in the test solution will result in the release of urease from the magnetic beads to the solution, which is then taken and used to hydrolyze urea in a reporter solution that contains a pH-sensitive dye. The final outcome of this procedure is a color change that can be conveniently reported by the dye or pH paper.

<table><tbody><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>VWR AMRESCO</td><td>0105</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH) pellets</td><td>BIO BASIC CANADA INC.</td><td>SB6789</td><td></td></tr><tr><td>Tris-base</td><td>VWR AMRESCO</td><td>0497</td><td></td></tr><tr><td>Boric acid</td><td>AMRESCO</td><td>0588</td><td></td></tr><tr><td>Urea</td><td>VWR AMRESCO</td><td>M123</td><td></td></tr><tr><td>40% acrylamide/bisacrylamide (29:1) solution</td><td>BIO BASIC CANADA INC.</td><td>A0007</td><td></td></tr><tr><td>Sucrose</td><td>Bioshop Canada inc.</td><td>SUC507</td><td></td></tr><tr><td>Bromophenol blue</td><td>Bioshop Canada inc.</td><td>BRO777</td><td></td></tr><tr><td>Xylenecyanol FF</td><td>SIGMA-ALDRICH</td><td>X-4126</td><td></td></tr><tr><td>10% sodium dodecyl sulfate</td><td>Bioshop Canada inc.</td><td>SDS001</td><td></td></tr><tr><td>Hydrochloric Acid (HCl)</td><td>CALEDON LABORATORIES LTD</td><td>6026</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Bioshop Canada inc.</td><td>SOD001</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Bioshop Canada inc.</td><td>HEP001</td><td></td></tr><tr><td>Magnesium Chloride (II) hexahydrate</td><td>VWR AMRESCO</td><td>0288</td><td></td></tr><tr><td>Tween 20</td><td>Bioshop Canada inc.</td><td>TW508</td><td></td></tr><tr><td>Adenosine Triphospahte (ATP)</td><td>AMRESCO</td><td>0220</td><td></td></tr><tr><td>Sodium Acetate trihydrate (NaOAc)</td><td>SIGMA-ALDRICH</td><td>S8625</td><td></td></tr><tr><td>Ethanol</td><td>Commercial Alcohols</td><td>P016EAAN</td><td></td></tr><tr><td>Tetramethyleneethylenediamine (TEMED)</td><td>AMRESCO</td><td>0761</td><td></td></tr><tr><td>10% Ammonium persulfate (APS)</td><td>BIO BASIC CANADA INC.</td><td>AB0072</td><td></td></tr><tr><td>Succinimidyl 4-(&lt;em&gt;N&lt;/em&gt;-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)</td><td>ThermoFisher SCIENTIFIC</td><td>22360</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>CALEDON LABORATORIES</td><td>803540</td><td></td></tr><tr><td>Urease</td><td>SIGMA-ALDRICH</td><td>U0251</td><td></td></tr><tr><td>1x Phosphate Buffered Saline (PBS)</td><td>ThermoFisher SCIENTIFIC</td><td>70011-069</td><td></td></tr><tr><td>0.04% Phenol red</td><td>SIGMA-ALDRICH</td><td>P3532</td><td></td></tr><tr><td>10x T4 polynucleotide kinase reaction buffer</td><td>Lucigen</td><td>30061-1</td><td></td></tr><tr><td>10x T4 DNA ligase reaction buffer</td><td>Bio Basics Canada</td><td>B1122-B</td><td></td></tr><tr><td>T4 DNA ligase (5 U/&amp;#956;l)</td><td>Thermo Fischer Scientific</td><td>B1122</td><td></td></tr><tr><td>Luria Bertani (LB) Broth</td><td>AMRESCO</td><td>J106</td><td></td></tr><tr><td>Agar</td><td>AMRESCO</td><td>J637</td><td></td></tr><tr><td>T4 polynucleotide kinase (10 U/&amp;#956;l)</td><td>Lucigen</td><td>30061-1</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; K12 (MG1655)</td><td>ATCC</td><td>ATCC700926</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter, Inc.</td><td>392187</td><td></td></tr><tr><td>Glass plates</td><td>CBS scientific</td><td>ngp-250nr</td><td></td></tr><tr><td>0.75 mm thick spacers</td><td>CBS scientific</td><td>VGS-0725r</td><td></td></tr><tr><td>12-well comb</td><td>CBS scientific</td><td>VGC-7512</td><td></td></tr><tr><td>UV Lamp</td><td>UVP</td><td>95-0017-09</td><td></td></tr><tr><td>Spectrophotometer (NanoVue)</td><td>GE Healthcare</td><td>N/A</td><td></td></tr><tr><td>Metal plate</td><td>CBS scientific</td><td>CPA165-250</td><td></td></tr><tr><td>Vortex</td><td>VWR International</td><td>58816-123</td><td></td></tr><tr><td>Gel electrophoresis apparatus</td><td>CBS scientific</td><td>ASG-250</td><td></td></tr><tr><td>Petri dishes</td><td>VWR International</td><td>25384-342</td><td></td></tr><tr><td>100 kDa MWCO centrifugal filters</td><td>EMD Millipore</td><td>UFC510024</td><td></td></tr><tr><td>Magnetic Bead (BioMag)</td><td>Bangs Laboratories Inc</td><td>BM568</td><td></td></tr><tr><td>Magnetic Seperation Rack</td><td>New England BioLabs</td><td>S1506S</td><td></td></tr><tr><td>Microfuge tubes</td><td>Sarstedt</td><td>72.69</td><td></td></tr><tr><td>Syringe filter (0.22 &amp;#956;m)</td><td>VWR International</td><td>28145-501</td><td></td></tr><tr><td>14 ml culture tube</td><td>VWR International</td><td>60818-725</td><td></td></tr><tr><td>Cell culture incubator</td><td>Eppendorf Scientific</td><td>M13520000</td><td></td></tr><tr><td>Branson Ultrasonic cleaner</td><td>Branson</td><td>N/A</td><td></td></tr><tr><td>Camera (Canon Powershot G11)</td><td>Canon</td><td>N/A</td><td></td></tr><tr><td>50 ml conical tube</td><td>VWR International</td><td>89004-364</td><td></td></tr></tbody></table>

colorimetric detection of bacteria using litmus test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

העיקרון של הנייר לקמוס חיידקי מוסבר באיור 1 המבחן משתמש בשלושה חומרי מפתח:. DNAzyme ביקוע-RNA כי הוא מופעל על ידי חיידק ספציפי, באוראז וחרוזים מגנטיים. DNAzyme משמש כאלמנט הכרה המולקולרי להשיג זיהוי ספציפי מאוד של חיידק עניין. באוראז וחרוזי מגנטים משמשים להשגה הולכים אותות של פעילות RNA-המחשוף של DNAzyme. זה כרוך ביצירת חרוזים מגנטיים המכילים conjugates באוראז-DNAzyme. בנוכחות של חיידק היעד, הצמדת RNA שלו ודבק DNAzyme. פעולה זו מעלה את הניתוק של באוראז מן החרוזים מגנטיים. באוראז שוחרר ניתן להפריד בקלות מן חרוזים מגנטיים רגילים לחולל שינוי צבע בתמיסה הכתב, המכיל אוריאה לצבוע pH רגיש. באוראז hydrolyzes אוריאה לתוך אמוניה, בליווי עלייה של pH שמפעיל את גשינוי olor של הצבע. איור 2 מציג מבחן לקמוס חיידקי שם EC1, א&#39; coli -responsive RNA-ביקוע DNAzyme, שימש DNAzyme, ו פנול אדום שימש לצבוע דיווח-pH. EC1 היה מבודד בעבר על ידי הקבוצה שלנו מתוך מאגר DNA אקראי רצף בטכניקה של בחירה במבחנה. 5 המחקרים הקודמים שלנו הראו כי EC1 הוא מאוד ספציפי עבור E. coli ותערוכות פעילות מינימלית כלפי חיידקים אחרים. 5,19 כבר נמצא כי EC1 מופעל על ידי מולקולת חלבון מן E. coli. למרות הזהות של סמן ביולוגי חלבון זה לא פוענחה, את הספציפיות הכרה הגבוהות עולה כי חלבון זה הוא ייחודי E. coli. פתרון הכתב מוגדר להיות בעל pH ראשונית של 5.5. ב- pH זה, פנול אדום מפגין צבע צהוב. כפי באוראז hydrolyzes אוריאה לתוך אמוניה, הבסיסית של סול הכתבעליות ution. הדבר בא לידי ביטוי על ידי השינוי ההדרגתי של צבע מצהוב ורוד. עומק השינוי בצבע תלוי בשני הפרמטרים הבאים, כפי שמודגם על ידי איור 2: מספר E. קולי תאים המשמשים שלב ההפעלה DNAzyme ואת הזמן המוקצב לשלב הידרוליזה אוריאה. עוד E. קולי תאי ביאה לשינויי צבע חזקים, המשתקפים התצפית של מעבר צבע צהוב-אל-ורוד מתקדם כאשר E. קולי תאים הוגדלו באופן סדרתי מן 5 5 x 10 7 (פי 10 להגדיל בכל פעם). בינתיים, זמן רב יותר עבור הידרוליזה אוריאה מותר לצורך זיהוי של מספרים קטנים של E. קולי תאים (5,000 תאים התגובה 1 שעה ו 500 התאים התגובה 2 שעות). השינוי pH של נייר לקמוס חיידקיים יכולים גם להיות במעקב באמצעות מד pH כף יד ותוצאות נציג הם באיור 3. WA זההים נמצא כי הנוכחות של 10 7 א קולי תאים הביאו לעלייה הדרגתית של pH על ידי 3 יחידות בתוך 10 דקות. לעומת זאת, בהיעדר E. קולי תאים לא לגרום לשינויים pH לזיהוי תחת אותה הגדרה. <img alt="איור 1" src="/files/ftp_upload/54546/54546fig1.jpg" /> איור 1: עיקרון העיצוב של נייר לקמוס בקטריאלי (א) הפעלה של DNAzyme ביקוע-RNA על ידי סמן ביולוגי ספציפי מחיידק של עניין.. בנוכחות של הסמן הביולוגי, את DNAzyme ביקוע-RNA משותק על מסלקת חרוזים מגנטית הצמדת RNA, וגורם לשחרור של באוראז מתויג מן החרוזים מגנטיים לפתרון. (ב) הליך assay שלושה שלבים. שלב 1: הפעלת DNAzyme, כמתואר בלוח א שלב 2: הפרדה מגנטי - באוראז שוחרר מופרד חרוזים מגנטיים. שלב 3: R הידרוליזה אוריאה12; באוראז שוחרר מתווסף לתוך פתרון כתב המכיל אוריאה. באוראז hydrolyzes אוריאה לתוך אמוניה, וכתוצאה מכך שינוי pH שניתן יהיה לדווח על ידי לצבוע pH רגיש. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54546/54546fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 2" src="/files/ftp_upload/54546/54546fig2.jpg" /> איור 2: נייר לקמוס עם E. coli באמצעות E. EC1 DNAzyme -responsive coli. נציג תוצאות צבע משתנה עם מספרים שונים של E. קולי תאים בתנאי מעל כל מבחנה. פנול אדום שימש לצבוע pH רגיש. מבחן ללא E. coli שימש כביקורת שלילית. עוד E. קולי תאים צפויים לגרום לשחרור מולקולות urease יותר, מלווה בy שינויי צבע חזקים. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54546/54546fig2large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 3" src="/files/ftp_upload/54546/54546fig3.jpg" /> איור 3: ניטור גידול pH באמצעות מד pH השינוי של pH נגרם על ידי 10 7 א. קולי תאים היה פיקוח באמצעות מד pH נייד. מבחן ללא E. coli שימש כביקורת שלילית. הנוכחות של 10 7 א coli תאים בתמיסת הבדיקה יכולים להגדיל את בסיסיות על ידי ~ 3 יחידות pH ב 10 דקות. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54546/54546fig3large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style=";text-align:right;direction:rtl"><tbody><tr><td> שֵׁם </td><td> Sequence (5&#39;-3 &quot;) </td><td> הערה </td></tr><tr><td> BS1 </td><td> BTTTT TTTTT TTTAC TCTTC CTAGC FRQGG TTCGA TCAAG </td><td> B: 5&#39;-ביוטין; R: ribonucleotide אדנין; F: והעמסה-DT; ש: dabcyl-DT </td></tr><tr><td> DE1 </td><td> GATGT GCGTT GTCGA GACCT GCGAC CGGAA CACTA CACTG TGTGG GGATG GATTT CTTTA CAGTT GTGTG TTGAA CGCTG TGTCA AAAAA AAAA </td><td></td></tr><tr><td> T1 </td><td> GACAA CGCAC ATCTC TTGAT CGAAC C </td><td></td></tr><tr><td> LD1 </td><td> XTTTT TTTTT TTTTT TTGAC ACAGC GTTCA </td><td> X: 5&#39;-NH 2 </td></tr></tbody></table> טבלה 1: רצפים של oligonucleotides הסינטטי.

Watch this Scientific Journal Video about Colorimetric Detection of Bacteria Using Litmus Test at JoVE.com

Colorimetric Detection of Bacteria Using Litmus Test

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

איתור מדד-צבע של חיידקים באמצעות מבחן לקמוס

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

colorimetric-detection-of-bacteria-using-litmus-test

Universidad San Sebastian

Research

JoVE Journal

Biology

9.1K Views.  McMaster University. We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads. 

Video: Colorimetric Detection of Bacteria Using Litmus Test

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

מבוסס נייר Colorimetric איתור של<em&gt; חיידקי Escherichia</em&gt;,<em&gt; סלמונלה</em&gt; Spp., ו<em&gt; חיידקי ליסטריה</em&gt; מכמויות גדולות של מים לחקלאות

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

Environment

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&quot;Bioluminescent&quot; כתב phage לצורך זיהוי של קטגוריה פתוגנים חיידקיים

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Immunology and Infection

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

גילוי חיידקים וזיהוי באמצעות חיישנים אלקטרוכימיים

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Bioengineering

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

זיהוי של חיידקים באמצעות DNAzymes Fluorogenic

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

שיטות מיקרוסקופית הקרינה לקביעת הכדאיות של חיידקים באיגוד עם תאי יונקים

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

ניטור שינויים בממברנה קוטביות, ממברנה יושרה, וריכוזי יון תאיים ב<em&gt; Streptococcus pneumoniae</em&gt; שימוש צבעי ניאון

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Visualizing Bacterial Motility Based on a Color Reaction

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

המחשת תנועתיות חיידקים בהתבסס על תגובת צבע

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

Water Quality Analysis via Indicator Organisms

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba -The University of Arizona 
Demonstrating Author: Luisa Ikner
Water quality analysis monitors anthropogenic influences such as pollutants, nutrients, pathogens, and any other constituent that can impact the water&#8217;s integrity as a resource. Fecal contamination contributes microbial pathogens that threaten plant, animal, and human health with disease or illness. Increasing water demands and strict quality standards require that water being supplied for human or environmental resources be monitored for low pathogen levels. However, monitoring each pathogen associated with fecal pollution is not feasible, as laboratory techniques involve extensive labor, time, and costs. Therefore, detection for indicator organisms provides a simple, rapid, and cost effective technique to monitor pathogens associated with unsanitary conditions.

Microbial Indicators and Most Probable Number Analysis for Water Quality

ניתוח איכות מים באמצעות אורגניזמים מחוון

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba -The University of Arizona
Demonstrating Author: Luisa Ikner
Water quality analysis monitors ...

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Environmental Microbiology

Photometric Protein Determination

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more a sample contains light-absorbing substances, the less the light will transmit through it. Since the relationship between concentration and absorption is linear, this phenomenon can be used to measure the concentration in samples where it is unknown.
This video describes the basics of photometric protein determination and introduces the Bradford Assay and the Lowry Method. The procedure in the video will cover a typical Bradford assay. Applications covered include direct measurement of very small volumes of nucleic acids to characterize concentration and purity, determination of coupling efficiency of a biomimetic material, and another variation of photometric protein determination using Remazol dye.
<div class="chapter_text" id="script_0">
	Determining the concentration of a protein in samples is a fundamental step in many biochemical assays. Photometric determination can be done with small sample sizes. The more a sample contains light-absorbing substances, the less the light will transmit through it. This provides a quantitative measurement of the absorbing substances. These concepts are so fundamental to science that the articles that introduced two of the techniques are in the three most cited papers of all time. This video will show the concepts behind some of the most common photometric protein determination techniques, how they are performed, and how the gathered data is analyzed.
</div>
<div class="chapter_text" id="script_1">
	Photometric protein determination is based on the relationship between concentration and light absorbency. This is known as the Beer-Lambert Law, which states that the concentration of a light-absorbing species is proportional to its absorbance.
	This principle underlies all photometric protein determination methods. 
	For direct absorption analysis, the absorbance values of unaltered protein samples are measured. Because of their aromatic side chains, tryptophan and tyrosine residues give the highest absorbance readings at a wavelength of 280 nm.
	However, these amino acids-which are two of the least frequently found in proteins-are present in different amounts in every protein, so each determination is unique. To overcome this limitation, more complex assays-that are not dependent on these amino acids-were developed.
	One example is the Bradford Assay, where colored dye is added to the sample. The dye, known as Coomassie Blue, responds proportionally-the more protein present, the more binding events with the dye.
	Then, protein concentration is determined by measuring the absorbance of the bound Coomassie Blue dye, which absorbs light at 594 nm. However, the Bradford assay is linear over a short range of concentrations, so dilutions are often required before analysis.
	The Lowry Method combines the Biuret reagent, an alkaline solution of copper ions that react with peptide bonds, and the Folin-Cioc&#226;lteu reagent, which oxidizes aromatic protein residues. The resulting color change of the sample is proportional to the protein concentration.
	The absorbance of the reduced Folin reagent can be determined at 750 nm. Like direct absorption, each protein has a unique response, and must be calibrated for the protein of interest. Now that we've reviewed the basic principles behind some of the most common assays, let's look at how direct absorption and the Bradford assay are performed.
</div>
<div class="chapter_text" id="script_2">
	To begin a direct absorption analysis, the spectrophotometer is calibrated with a blank to determine zero absorbance. Standard solutions are prepared for use in creating the calibration curve. Then, an aliquot of the first standard is added to a cuvette, and placed into the spectrophotometer.
	The absorbance value at 280 nm is then recorded. This process is repeated for each standard, using a clean cuvette for each run. Once complete, a calibration curve is created by plotting the absorbance versus concentration. The slope of this line is the molar attenuation coefficient, which relates absorbance to concentration.
	Next, the unknown sample is added to a cuvette, and the absorbance value is recorded. As the data analysis for the different photometric determination methods is similar, we will cover that after we look at the Bradford assay.
	Here, the Bradford protein assay is performed with a BSA standard on a 96-well plate. To begin, BSA stock solutions are prepared.
	The unknown solutions are diluted with deionized water to ensure that the concentrations are within the assay's range. Depending on the kit, the Coomassie dye may also require dilution. Then, the calibration curve is set up by adding the BSA standards to the 96-well plate.
	Deionized water is added to reach the needed concentration to generate a standard curve. The unknown sample should be added to the plate in triplicates to ensure an accurate measurement is taken. Coomassie dye is next added to each well, mixing with the pipette.
	Deionized water is added to an empty well as a blank, to measure the absorbance. After waiting 5 min for the dye to bind, the absorbance is measured in a plate-reader at 590 nm.
</div>
<div class="chapter_text" id="script_3">
	Now that we've performed a few assays, let's look at how to analyze the data. Each photometric protein determination method is based on the Beer-Lambert Law.
	The measured absorbance of the standards is used to create a calibration curve, which is then used to determine the concentration of unknown samples. This curve can be manually plotted, though newer spectrophotometric tools will create the calibration curve once all standards have been measured. These systems will also calculate protein concentration as unknown samples are analyzed.
</div>
<div class="chapter_text" id="script_4">
	Now that we've reviewed how to analyze photometric protein determination data, let's look at some of the ways these procedures are utilized.
	The principles of photometric protein determination can also be used to directly measure nucleic acid concentration. The nanodrop spectrophotometer accepts samples of very small volume onto an optically active pedestal. The absorbance is then measured, and the system automatically determines the nucleic acid concentration. Because proteins and other sources can interfere with measurements, sample purity is determined by analyzing the 260 to 280 nm and 260 to 230 nm absorbance ratios. Pure nucleic acids typically yield ratios of approximately 1.8 and approximately 2.0 for DNA and RNA, respectively.
	Photometric protein determination can also be used in the production of biomimetic materials, which are inspired from nature to elicit specific cellular responses. Recombinant adhesins are bound to polystyrene beads to simulate bacterial attachment to host cells. The Bradford assay is used to determine the coupling efficiency of the recombinant adhesion to the beads in the production of the biomimetic material.
	Alternative photometric protein assays can be used in the detection and characterization of protein antimicrobials. Remazol brilliant blue R dye is covalently bonded to heat-killed bacteria. The protein antimicrobial is incubated in the dyed solution. Then, the sample is centrifuged, and the absorbance of the supernatant at 595 nm is measured using a microplate spectrophotometer. Increased absorbance, by the soluble dye released into the supernatant from the labeled bacteria, is a quantitative measurement of enzymatic activity.
</div>
<div class="chapter_text" id="script_5">
	You've just watched JoVE's video on photometric protein determination. This video described the underlying principles of photometric determination, went over general procedures for some common assays, and covered some new advances in techniques. Thanks for watching!
</div>

Photometric Protein Determination: Bradford Assay

קביעת חלבונים פוטומטריים

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more ...

Chemistry

Biochemistry

Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test

After preparing E. coli cells according to the text protocol, pre-wash a 1.5-milliliter microfuge tube by adding and vortexing 100 microliters of RB. Then, discard the buffer.
Add 15 microliters of assembled EC1 to the washed tube, then, place the tube on the magnetic rack, and aspirate the supernatant. Remove the tube from the rack, add 100 microliters of RB, and carefully resuspend the magnetic beads.
After using RB to wash the beads two more times and removing the RB, add 10 microliters of the E. coli sample to the tube, and gently mix the contents by tapping on the tube. Then, incubate the reaction at room temperature for one hour.
After the incubation, add 90 microliters of doubly-distilled water, and place the tube on the magnetic rack. Following approximately three minutes of magnetic separation, carefully transfer 85 microliters of the supernatant to a 0.5-milliliter microfuge tube.
It is very important to pipette the solution carefully without taking up any of the magnetic beads. These beads are loaded with urease and will generate a false positive signal.
Add 15 microliters of 0.04% phenol red and 100 microliters of substrate solution. Then, take photographs at specific time intervals to record a color change.

איתור מדד-צבע של חיידקים באמצעות מבחן לקמוס

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מבוסס נייר Colorimetric איתור של

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