The author thanks R. Boss, A. Cosandey, C. Fournier, I. Ivanovic, and J. Naskova for their excellent work.

1. المكورات العنقودية الذهبية العزلات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نشر 10 ميكرولتر من س. الذهبية ثقافة الأسهم أو مستعمرة واحدة نمت على وسيلة انتقائية على لوحات أجار كولومبيا تحتوي على 5٪ الدم الأغنام (BA). </li><li style=";text-align:right;direction:rtl"> احتضان هوائيا على 37 درجة مئوية لمدة 18 ساعة إلى 24 ساعة. </li></ol> 2. استخراج الحمض النووي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد عازلة هاتف تحتوي على 10 ملي تريس، حمض الهيدروكلوريك و 10 ملي نا 2 EDTA، ودرجة الحموضة 8.5. إعداد قسامات والأوتوكلاف على 121 درجة مئوية لمدة 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> جمع 1 مستعمرة من BA باستخدام حلقة عقيمة البلاستيك أو اختيار الأسنان الخشبي المعقم و resuspend في 100 هاتف ميكرولتر. استخدام 1.5 مل أنابيب مع قبعات المسمار. </li><li style=";text-align:right;direction:rtl"> احتضان عند 95 درجة مئوية لمدة 10 دقيقة. بعد ذلك، ضع عينات فورا على الجليد الرطب. </li><li style=";text-align:right;direction:rtl"> تمييع عينات 1: 100 في H 2 O مناسبة لPCR (= قالب الحمض النووي للPCR). يمكن حفظ العينات غير المخفف والمخفف في -20 درجة مئوية لمدة 1 شهر. </li></ol> 3. RS-PCR <ol style=";text-align:right;direction:rtl"><lط&gt; لكل ماصة العينة إلى PCR أنبوب 1.5 ميكرولتر H 2 O، 12.5 ميكرولتر طق ميكس ماستر، 2 ميكرولتر G1 التمهيدي 10 ميكرومتر، 2 ميكرولتر التمهيدي L1 10 ميكرومتر، و 7 ميكرولتر من الحمض النووي القالب الذي يتوافق مع حوالي 30 نانوغرام من الذهب الخالص الحمض النووي (2). </li><li style=";text-align:right;direction:rtl"> تشغيل برنامج الدراجات التالية في cycler على PCR القياسية: 15 دقيقة 95 درجة مئوية، تليها 27 دورات تضم 94 درجة مئوية لمدة 1 دقيقة، تليها منحدر 2 دقيقة والصلب في 55 درجة مئوية لمدة 7 دقائق. بعد 2 دقيقة منحدر آخر، تمديد عند 72 درجة مئوية لمدة 2 دقيقة. إنهاء PCR بواسطة ملحق النهائي عند 72 درجة مئوية لمدة 10 دقيقة تليها تهدئة إلى 4 درجات مئوية. ملاحظة: منتجات PCR يمكن تخزينها في -20 درجة مئوية لمدة 5 أيام. </li><li style=";text-align:right;direction:rtl"> لكل شوط من RS-PCR تشمل مراقبة إيجابية، أي عينة مع النمط الجيني المعروف (عادة GTB)، والسيطرة السلبية (H 2 O). </li></ol> 4. الكهربائي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام عدة الكهربائي المنمنمة التجارية لالحمض النووي (مجموعة MED) معامع bioanalyzer متصلة بجهاز كمبيوتر والبرامج المثبتة. دراسة الأدلة المقابلة من الشركة المصنعة بعناية. </li><li style=";text-align:right;direction:rtl"> إعداد محطة فتيلة رقاقة وضبط مقطع حقنة وفقا لدليل من الشركة المصنعة. </li><li style=";text-align:right;direction:rtl"> تحضير مزيج هلام صباغة وفقا لدليل من الشركة المصنعة. </li><li style=";text-align:right;direction:rtl"> تحميل مزيج هلام صباغة (وفقا لدليل من الشركة المصنعة). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تتوازن مزيج هلام الصبغة لRT لمدة 30 دقيقة قبل الاستخدام. </li><li style=";text-align:right;direction:rtl"> وضع رقاقة الحمض النووي الجديد على محطة رقاقة فتيلة. ماصة 9 ميكرولتر من هلام الصبغة في G. جيدا ملحوظ </li><li style=";text-align:right;direction:rtl"> تأكد من أن يتم وضع المكبس في 1 مل ثم قم بإغلاق محطة رقاقة فتيلة. الصحافة المكبس حتى يتم عقد من قبل مقطع حقنة. انتظر بالضبط لمدة 30 ثانية ثم حرر مقطع حقنة. </li><li style=";text-align:right;direction:rtl"> الانتظار لمدة 5 ثوان. سحب ببطء مرة أخرى المكبس إلى 1 مل الموقف. </li><li style=";text-align:right;direction:rtl"> فتح رقاقة محطة فتيلة وماصة 9 ميكرولتر من هلام الصبغة في كل من الآبار ملحوظ &#39;G&#39;. </li></ol></li><li style=";text-align:right;direction:rtl"> تحميل Marker <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ماصة 5 ميكرولتر من علامة في جميع الآبار عينة 12 وفي سلم أيضا. لا تترك أي من هذه الآبار 13 فارغة. </li></ol></li><li style=";text-align:right;direction:rtl"> تحميل سلم والعينات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ماصة 1 ميكرولتر من سلم ال DNA إلى تميز جيدا مع علامة سلم. </li><li style=";text-align:right;direction:rtl"> ماصة 1 ميكرولتر من عينة (الآبار المستخدمة) أو 1 ميكرولتر من الماء منزوع الأيونات (الآبار غير المستخدمة). </li><li style=";text-align:right;direction:rtl"> وضع رقاقة أفقيا في محول ودوامة لمدة 1 دقيقة في 2400 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> تشغيل رقاقة في bioanalyzer خلال 5 دقائق. </li></ol></li><li style=";text-align:right;direction:rtl"> تشغيل تحليل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بدء bioanalyzer وجهاز كمبيوتر متصل. فتح البرنامج. </li><li style=";text-align:right;direction:rtl"> فتح غطاء bioanalyzer ووضع رقاقة في حامل رقاقة (شريحة يناسب طريقة واحدة فقط). إغلاق الغطاء بعناية. </li><li style=";text-align:right;direction:rtl"> في سياق صك البرنامج حدد &quot;الكهربائي&quot;&gt; &quot;dsDNA&quot;&gt; &quot;DNA 7500&quot;. استعرض بادئة الملف الحالي من البرنامج أو تعديله.</li><li style=";text-align:right;direction:rtl"> انقر على زر البداية موجودة في البرنامج. أدخل أسماء عينة في البرنامج. عندما يتم الانتهاء من تشغيل رقاقة (بعد حوالي 30 دقيقة) يظهر نهاية الرسالة تشغيل في البرنامج bioanalyzer. </li><li style=";text-align:right;direction:rtl"> إزالة الشريحة وتنظيف الأقطاب من bioanalyzer وفقا لتعليمات الشركة المصنعة. </li></ol></li></ol> 5. استنتاج التركيب الوراثي من الكهربائي الملف الشخصي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحميل البرنامج محل (البرنامج متاح بحرية من المؤلفين). </li><li style=";text-align:right;direction:rtl"> بيانات الاستيراد إشارة إلى البرنامج محل: &quot;ملف&quot;&gt; &quot;استيراد بيانات المرجع&quot; </li><li style=";text-align:right;direction:rtl"> فتح الكهربائي تشغيل في سياق البيانات من البرنامج bioanalyzer. حدد ملف تعريف الكهربائي من عينة واحدة. </li><li style=";text-align:right;direction:rtl"> تحقق من القمم ذات الصلة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام البرنامج محل للتحقق من القمم ذات الصلة: &quot;أدوات&quot;&gt; &quot;يعني الإسفار&quot;. أدخل في مربع &quot;الإسفار&quot;وأشارت القيم مضان وحدات مضان (FU) لأنها يمكن أن تقرأ من برنامج bioanalyzer. إدراج 4 (3) قمم مع أعلى مضان في ترتيب تصاعدي ومفصولة بفواصل. مثال: 126130132137. ملاحظة: إذا لم يكن كل هذه القمم FU بالرمز البرنامج bioanalyzer، فإنها تحتاج إلى أن يقدر من خلال قراءة لهم للخروج من هذه المؤامرة. </li></ol></li><li style=";text-align:right;direction:rtl"> اضغط على زر &quot;حساب&quot;. الذروة هي ذات الصلة إذا تجاوز مضان احظ قيمة معينة في مربع &quot;ذروة ارتفاع الحد الأدنى&quot;. </li><li style=";text-align:right;direction:rtl"> استدلال على التركيب الوراثي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إدراج في محل النص &quot;قمم الإدخال (بي بي)&quot; أحجام في بي بي من كل القمم ذات الصلة في ترتيب تصاعدي ومفصولة بفواصل. على سبيل المثال: 440.462.519.579.637. ملاحظة: إذا يشار ليس كل من هذه الأحجام الذروة من قبل البرنامج bioanalyzer، فإنها تحتاج إلى أن يقدر من خلال قراءة لهم للخروج من هذه المؤامرة. </li></ol></li><li style=";text-align:right;direction:rtl"> اضغط على زر &quot;حساب&quot;. </li><li style=";text-align:right;direction:rtl"> في القائمةمربع، والبحث عن الحد الأدنى المربعة مسافة Mahalanobis &quot;D2M&quot;. إذا كانت قيمة P المقابلة الحق في ذلك هو ≥0.05 ثم احتمال هو ≥5٪ أن فرضية H باطل 0 تم رفض (H 0: لوحظ والتعريف إشارة متطابقة) وهذا يعني أن الشخصية احظ قبلت أن تكون مطابقة ل البيانات الشخصية المرجعية من النمط الجيني المشار إليه. </li><li style=";text-align:right;direction:rtl"> إذا D2M هو الحد الأدنى لكن الحقل من قيمة P المقابلة فارغة، رفض H 0 مع وجود احتمال &lt;5٪. ملاحظة: هذا يعني أن الشخصية هي غير متكافئة على الرغم من D2M هو الحد الأدنى. وقد يؤدي مثل هذه النتيجة من غير الظروف العادية الكهربائي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لتصحيح هذه الانحرافات، أدخل في مربع النص &quot;لوحظ الذروة (بي بي)&quot; قيمة 395 واضغط على زر &quot;حساب&quot;. التحقق مرة أخرى لأدنى D2M و≥0.05 قيمة P المقابلة. إذا لم يكن ناجحا، حاول القيم 405، 415، و 425. ملاحظة: في بعض الأحيان خطوات أصغر، بل هيالمشار إليها. إذا كان لا يزال لم تكن ناجحة، والوضع احظ غير موجود في البيانات المرجعية، قد يكون من النمط الجيني الجديد أو البديل. </li></ol></li><li style=";text-align:right;direction:rtl"> لمحات مختلفة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> كرر الخطوات من 5،6-5،9 لكل ملف على حدة الكهربائي. </li></ol></li></ol>

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The authors have nothing to disclose.

الخطوة الأكثر أهمية الإجراء كله هو عندما يكون هناك فائض من الحمض النووي في RS-PCR (&gt; 30 نانوغرام النقي DNA / الفحص) 2. بشكل عام، قرار من هذا التوصيف الأمثل إذا كان كل من مستويات الذروة التي بلغتها تبدأ وتنتهي عند خط الأساس. إذا قمتين متقاربة جدا، والقرار هو غير مكتمل. ف?...

المكورات العنقودية (S. المذهبة) كما هو معروف الممرض أهم المسؤولين في جميع أنحاء العالم للعدوى داخل الثدي (IMI) في الماشية 1. الدراسة التي فورنييه وآخرون. 2 موضح في الأبقار السويسرية والدراسات التي Cosandey وآخرون. (3) وبوس وآخرون. 4 في الأبقار من 12 دولة أوروبية أن س. العنقودية الذهبية المعزولة من الأبقار IMI هي مجموعة غير متجانسة وراثيا. بواسطة PCR التضخيم من 16S-23S الريباسي المنطقة فاصل بين الجينات (RS-PCR)، تم الكشف عن ما مجموعه 17 المورثات في البداية في 101 العزلات مستقلة وبائيا 2. كان النمط الجيني B و C التركيب الوراثي (GTC) الأكثر شيوعا (80٪)، في حين أن الأخرى 15 المورثات (GTS) وقعت نادرا، مما يجعل كل 1-4٪ من العزلات. على المستوى الأوروبي 3، GTB، GTC، GTF، كان GTI، وGTR التراكيب الوراثية أهم تضم 76٪ من جميع سلالات تحليلها (ن = 456). كانت تقع GTB في وسط أوروبا ثhereas وزعت على نطاق واسع في المورثات الأخرى. شملت 24٪ المتبقية من سلالات 41 المورثات، وكثير منهم، ومع ذلك، لوحظت مرة واحدة فقط. أثبتت الدراسات التي فورنييه وآخرون. 2، Cosandey وآخرون. 3، وجرابر وآخرون. 5 أيضا أن المورثات كانت مرتبطة إلى حد كبير مع نمط الفوعة الجينات الخاصة بهم، وكذلك في سويسرا بصفتها ممثلة على المستوى الأوروبي. كشف مزيد من الدراسات اختلافات كبيرة في انتشار IMI بين س. أصيب النظر GTB، تصل إلى 87٪ من الأبقار في قطيع من هذا النمط الجيني: الذهبية GTB، GTC والمورثات الأخرى 2،5. في حالة GTC ومن المورثات الأخرى، ومع ذلك، IMI تم العثور فقط في 1-2 الأبقار من قطيع. IMI التي تسببها س. الذهبية، يؤدي عادة إلى التهاب في الغدد الثديية المقابلة وبزيادة الخلايا الالتهابية في الحليب. ونتيجة لذلك، وشارك الخلايا الجسديةUNTS في الحليب (SCC)، المؤشر الرئيسي للجودة الحليب في معظم البلدان، وتزداد مما أدى إلى سعر الحليب انخفضت أو حتى توقف التسليم. إلى جانب RS-PCR من فورنييه وآخرون. 2، وقد وصفت أساليب الكتابة الأخرى لعملية التصنيف الفرعي لسلالات الأبقار من S. الذهبية 8-11. تشمل اثنين من تلك الشائعة في الكتابة سبا 12 و متعدد الحالة رقم تسلسل الكتابة (MLST) 13. ويستند السابق واحد على تسلسل الحمض النووي في المنطقة هل المتغيرة من الجين الترميز سبا العنقودية للبروتين A، في حين أن واحدة الأخير يتطلب تسلسل 7 جينات التدبير المنزلي. هذا يعني أن واحدا 12 وسبعة تقارير إنجاز المشروعات 13، على التوالي، في حاجة إلى أن يقوم، تليها تنقية amplicon، رد فعل التسلسل والتحليل باستخدام معدات معينة. مقارنة RS-PCR، وهذه الأساليب تتطلب جهدا إضافيا كبيرا في المختبر مما أدى إلى سرعة عينة انخفاض وارتفاع التكاليف. الإنتاجية منخفضة بشكل خاص لPFGE. وبالنظر إلى قرار من هذه الأساليب لسلالات الأبقار من S. الذهبية، RS-PCR على الأقل جيدة كما سبا كتابة 4 وأفضل من MLST 4 أو PFGE 15. أظهرت جميع هذه الطرق بما في ذلك الكتابة الثنائية 13 فعاليتها في الحصول على مزيد من التبصر في التسبب في الأبقار IMI التي تسببها س. الذهبية، ولكن بسبب القيود المذكورة هم أقل ملاءمة للتحقيقات سريرية واسعة النطاق. وبالإضافة إلى ذلك، التنميط الجيني عن طريق RS-PCR كما وصفها فورنييه وآخرون. 2 يسمح للتنبؤ موثوق لالوبائية واحتمال المسببة للأمراض من س. الذهبية تشارك في الأبقار IMI، قيمتين الرئيسية في الطب البيطري السريري.

<table><tbody><tr><td>Tris(hydroxymethyl)-aminomethan</td><td>Merck</td><td>1.08382.0500</td><td>Mw = 121.14 g/mol</td></tr><tr><td>Titriplex III</td><td>Merck</td><td>1.08418.0250</td><td>Mw = 372.24 g/mol; Substance is equivalent to Na&lt;sub&gt;2&lt;/sub&gt;EDTA&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Hydrochloric acid 5 mol/L</td><td>Merck</td><td>1.09911.0001</td><td></td></tr><tr><td>Columbia agar+5% sheep blood</td><td>bioM&amp;eacute;rieux</td><td>43049</td><td></td></tr><tr><td>Agilent DNA7500 Kit</td><td>Agilent Technologies</td><td>5067-1504</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2940CA</td><td></td></tr><tr><td>Agilent 2100 expert sofware</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>HotStarTaq master mix&amp;nbsp;</td><td>Qiagen</td><td>203445</td><td></td></tr><tr><td>Primer L1</td><td>Mycrosinth</td><td></td><td>5&#039;CAA GGC ATC CAC CGT3&#039;</td></tr><tr><td>Primer G1</td><td>Mycrosinth</td><td></td><td>5&#039;GAA GTC GTA ACA AGG3&#039;</td></tr></tbody></table>

genotyping of staphylococcus aureus by ribosomal spacer pcr (rs-pcr)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

تعريف النمط الجيني ومشتقاته ويعتبر ملف تعريف الكهربي مختلفة في الفرقة أكثر من واحد من كل المورثات التي تم تحديدها باعتبارها النمط الجيني الجديد. يتم تسمية المورثات جديدة وتمديد وفقا لفورنييه ...

Watch this Scientific Journal Video about Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR at JoVE.com

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

التنميط الجيني ل<em&gt; المكورات العنقودية الذهبية</em&gt; من جانب خاص بالريبوسوم فاصل PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

genotyping-of-staphylococcus-aureus-by-ribosomal-spacer-pcr-rs-pcr

Research

JoVE Journal

Immunology and Infection

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

9.6K Views.  Agroscope. Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

Video: Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

تقييم التلوث الحمض النووي في عينات الحمض النووي الريبي استناداً إلى ريبوسومال الحمض النووي

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Genetics

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

جيل تعطيل الجينات العقدية mutans سلالة دون الجيني الاستنساخ

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

جهاز الاستشعار البيولوجي للكشف عن المضادات الحيوية البكتيريا العنقودية الذهبية المقاومة لل

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Bioengineering

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.

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

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

MS2-تقارب تنقية مقرونة تسلسل الحمض النووي الريبي في البكتيريا إيجابية الغرام

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

Biochemistry

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

تحديد مسببات الأمراض البكتيرية النادرة التي 16S الريباسي جين تسلسل وMALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

حذوفات الجينات في الجينوم على نطاق<em&gt; العقدية الدم</em&gt; من جانب PCR إنتاجية عالية

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Biology

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

الجيل المقبل من تسلسل خاص بالريبوسوم 16S RNA جين Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

كفاءة نوكليك حمض استخراج و16S الريباسي جين تسلسل للبكتيريا الجماعة توصيف

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

تحليل الكائنات المجهرية باستخدام ثنائي الخطوة PCR والجيل التالي من 16S rRNA التسلسل الجيني

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

التنميط الجيني ل

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