Name: genotyping of staphylococcus aureus by ribosomal spacer pcr (rs-pcr)
Uploaded: 2016-11-04T13:00:00.000+00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR at JoVE.com

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

1. Staphylococcus aureus izolatları <ol><li> S. 10 ul yayıldı aureus hazır bir kültür veya bir koloni,% 5 koyun kanı (BA) içeren Kolumbiya agar plakaları üzerinde seçici bir ortam üzerinde büyütülür. </li><li> 24 saat için 18 saat, 37 ° C &#39;de aerobik inkübe edin. </li></ol> 2. DNA Ekstraksiyon <ol><li> 10 mM Tris-HCl ve 10 mM Na2EDTA, pH 8.5 ihtiva eden TEl tamponu hazırlayın. 15 dakika boyunca 121 ° C&#39;de alikotları ve otoklav hazırlayın. </li><li> 100 ul TEL steril plastik döngü veya steril ahşap kürdan ve tekrar süspansiyon kullanılarak BA 1 koloni toplayın. vidalı kapakları ile 1.5 ml tüpler kullanın. </li><li> 10 dakika boyunca 95 ° C&#39;de inkübe edin. Daha sonra, ıslak buz üzerinde hemen örnekleri yerleştirin. </li><li> PCR (PCR için = şablon DNA) için uygun H 2 O 100: numuneleri 1 seyreltin. olmayan seyreltildi ve seyreltilmiş numune, 1 ay boyunca -20 ° C&#39;de muhafaza edilebilir. </li></ol> 3. RS-PCR <ol><lPCR tüpü 1.5 ul H içine her bir numune pipet için i&gt; 2 O, Taq Master Mix ul 12.5, 2 ul G1 astar 10 uM, 2 ul L1 astar 10 uM ve saf yaklaşık 30 ng karşılık gelen şablon DNA 7 ul DNA, 2. </li><li> Standart bir PCR döngü aşağıdaki döngü programı çalıştırmak: 15 dakika, 7 dakika boyunca 55 ° C &#39;de 2 dakika rampa ve tavlama takip 1 dakika için 94 ° C kapsayan 27 çevrim, ardından 95 ° C. Bir başka 2 dakika rampa sonra 2 dakika süreyle 72 ° C&#39;de uzatma. 4 ° C&#39;ye kadar soğutulduktan takiben 10 dakika boyunca 72 ° C&#39;de son uzatma PCR sonlandırma. Not: PCR ürünleri, 5 gün boyunca -20 ° C&#39;de saklanabilir. </li><li> Her çalışma için, RS-PCR, yani pozitif kontrol, bilinen bir genotip (normalde GTP) bir örneği, ve bir negatif kontrolü (H2O). </li></ol> 4. Elektroforez <ol><li> Birlikte DNA (MED kiti) için bir ticari minyatür elektroforez kiti kullanınPC ve yüklü yazılım bağlı bir Bioanalyzer ile. dikkatle üreticinin gelen kılavuzları inceleyin. </li><li> çip astar istasyonu kurmak ve üreticinin kılavuzuna göre şırınga klibi ayarlayın. </li><li> üreticinin kılavuzuna göre jel boya karışımı hazırlayın. </li><li> (Üreticinin kılavuzuna göre) jel boya karışımı yükleniyor. <ol><li> kullanımdan önce, 30 dakika boyunca oda sıcaklığında jel boya karışımı dengelenmesi. </li><li> Çip astar istasyonunda yeni bir DNA çip koyun. iyi işaretlenmiş G. jel boya Pipet 9 ul </li><li> piston 1 ml konumlandırılmış olduğundan emin olun ve sonra çip hazırlama istasyonu kapatın. Basın piston o şırınga klibi tarafından tutulan kadar. 30 sn sonra şırınga klibi yayınlayacak için tam bekleyin. </li><li> 5 saniye bekleyin. Yavaşça 1 ml konumuna pistonu geri çekin. </li><li> Her iki kuyularda Açık çip hazırlama istasyonu ve jel boya pipet 9 ul &#39;G&#39; olarak işaretlenmiş. </li></ol></li><li> M Yüklemearker <ol><li> 12 örnek kuyularda ve iyi merdivende marker Pipet 5 ul. Boş olan bu 13 kuyu herhangi bırakmayın. </li></ol></li><li> Merdiven ve Örnekleri yükleniyor. <ol><li> Pipet merdiven işareti ile iyi işaretlenmiş DNA merdiveninin 1 ul. </li><li> Numunenin Pipet 1 ul (kullanılan kuyular) ya da deiyonize su (kullanılmayan kuyuların) 1 ul. </li><li> 2400 rpm&#39;de 1 dakika için adaptör ve vorteks yatay çip koyun. </li><li> 5 dakika içinde Bioanalyzer çipi çalıştırın. </li></ol></li><li> Analizi Koşu <ol><li> Bioanalyzer ve bağlı bilgisayarınızı başlatın. yazılımını açın. </li><li> Bioanalyzer kapağını açın ve (çip yalnızca bir şekilde oturabilir) yonga tutucu içine çip yerleştirin. dikkatlice kapağını kapatın. </li><li> Yazılımın Enstrüman bağlamında &quot;Elektroforez&quot;&gt; &quot;dsDNA&quot;&gt; &quot;DNA 7500&quot; i seçin. Yazılımın güncel Dosya Öneki kabul edin veya değiştirin.</li><li> yazılımda mevcut start düğmesine tıklayın. Yazılımın örnek adlarını girin. Çip çalışma bittiğinde (sonra yaklaşık 30 dakika) Run mesajın sonu Bioanalyzer yazılımında görünür. </li><li> üreticinin talimatlarına göre Bioanalyzer elektrotları çipini çıkarın ve temizleyin. </li></ol></li></ol> 5. Elektroforez Profilinden genotip çıkarımlar yapma <ol><li> (Yazılım yazarlardan serbestçe kullanılabilir) Mahal yazılımını yükleyin. </li><li> &gt; &quot;Import Referans Veri&quot; &quot;Dosya&quot;: Mahal yazılımı içine alma referans verileri </li><li> Bioanalyzer yazılımı Veri bağlamında çalışır elektroforez açın. Bir numunede bir elektroforez profili seçin. </li><li> İlgili zirveleri için kontrol edin. <ol><li> &quot;Araçlar&quot;&gt; &quot;Mean Floresans&quot;: İlgili zirveleri kontrol etmek için Mahal yazılımını kullanın. &quot;Floresan&quot; kutusuna yerleştirinfloresans değerleri, Bioanalyzer yazılımından okunabilir şekilde, floresans (FU) olarak gösterilir. artan düzende en yüksek floresan 4 (3) zirveleri takın ve virgülle ayrılmış. Örnek: 126.130.132.137. NOT: tüm bu zirveleri FU Bioanalyzer yazılımı ile gösterilir, bunlar komplo onları okuyarak tahmin edilmesi gerekmektedir. </li></ol></li><li> &quot;Hesapla&quot; tuşuna basınız. gözlenen floresan kutusuna &quot;Minimal pik yüksekliği&quot; verilen değeri aştığı takdirde bir zirve geçerlidir. </li><li> genotip anlaması. <ol><li> Mahal metin kutusu &quot;Giriş Peaks (bp)&quot; artan sırayla ilgili tüm piklerin bp ölçü ve virgülle ayrılmış içine yerleştirin. Örnek: 440.462.519.579.637. NOT: Bu en yüksek boyutlarda tüm Bioanalyzer yazılımı ile gösterilir, bunlar grafiğinden onları okuyarak tahmin edilmesi gerekmektedir. </li></ol></li><li> &quot;Hesapla&quot; tuşuna basınız. </li><li> listesindekutu, asgari kare Mahalanobis mesafe &quot;d2m&quot; arayın. Bu ≥0.05 sağ karşılık gelen p değeri daha sonra olasılık sıfır hipotezi H 0 reddedilir ≥5% ise (H 0: gözlenen ve referans profili aynıdır) gözlenen profilini özdeş olduğu kabul edilmektedir, yani belirtilen genotip referans profili. </li><li> D2m az ancak karşılık gelen P değerinin alan boş ise, bir olasılık &lt;% 5 ile H 0 reddediyoruz. Not: Bu d2m çok az olsa da profilleri eşit olduğu anlamına gelir. Bu tür bir sonuç, sigara ortalama elektroforez koşulları kaynaklanabilir. <ol><li> Bu sapmaların düzeltmek için, değeri 395 &quot;Tepe (bp) Gözlenen&quot; metin kutusuna takın ve &quot;Hesapla&quot; tuşuna basınız. Minimal d2m ve karşılık gelen P değeri ≥0.05 için tekrar kontrol edin. Başarılı değilse, değerleri 405, 415, ve 425 deneyin. NOT: Bazen küçük adımlar bile vardırbelirtilen. Gözlenen profil Referans veri mevcut değildir başarılı de, bu yeni genotip veya varyant olabilir. </li></ol></li><li> çeşitli Profiller <ol><li> Yineleyin ayrı ayrı 5.6 her elektroforez profili için 5.9 için yineleyin. </li></ol></li></ol>

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The authors have nothing to disclose.

RS-PCR (&gt; 30 saf DNA / tahlil ng) &#39;de DNA&#39; sının aşırı olduğunda bütün prosedürün en kritik adımdır 2. Bunu tüm tepe başlangıçta başlangıç ​​ve bitiş Genel olarak, bir profilin çözünürlüğü en uygunudur. iki tepe birbirine çok yakın ise, çözünürlük eksik. kılavuzu tanımlama gerekli olduğu şekilde, bu koşullar altında, Bioanalyzer yazılımı olmayan pik tanımlanmasına yol açabilir. bp veya FU olarak ifade edilen tüm gözle ...

Staphylococcus (S. aureus) sığır 1 meme içi enfeksiyon (IMI) dünya çapında sorumlu en önemli patojen olarak bilinir. 12 Avrupa ülkesinde bunun ineklerde Cosandey ve ark., 3 ve Boss ve diğ. 4 tarafından Fournier ve ark. 2 İsviçre ineklerde gösterdi ve çalışmalarla çalışma S. aureus IMI genetik olarak heterojen bir grup büyükbaş izole. 16S-23S rRNA intergenik aralayıcı bölge (RS-PCR), PCR amplifikasyonu ile, 17 genotip toplam başlangıçta 101 epidemiyolojik bağımsız izolatların 2 tespit edilmiştir. Diğer 15 genotipleri (GTs) her 1 tüm izolatların% 4, yapım nadiren meydana oysa Genotip B ve genotip C (GTC), (% 80) en sık görülen idi. Avrupa 3. seviyede, GTB, GTC, GTF, GTI ve GTR tüm analiz suşları (n = 456)% 76 içeren en önemli genotipleri vardı. GTB w Orta Avrupa&#39;da yer alıyorduhereas diğer genotip yaygın dağıtılmıştır. Suşların kalan% 24 birçoğu, ancak, sadece bir kez gözlendi 41 genotipleri dahil. Genotipleri çok Avrupa düzeyinde olduğu Swiss&#39;te yanı sıra, onların virülans gen modeli ile ilişkili olduğunu Fournier ve ark. 2, Cosandey vd. 3 ve Graber vd. 5 ile çalışmalar daha gösterdi. Çalışmalar daha S. arasında IMI prevalansının büyük farklılıklar ortaya aureus GTB, GTC ve diğer genotipleri 2,5: GTB dikkate alınarak, bir sürüdeki ineklerin% 87&#39;ye kadar bu genotip ile enfekte edildi. GTC ve diğer genotiplerinin durumda, ancak, IMI sadece sürünün 1 ila 2 ineklerde tespit edildi. IMI S. neden aureus, normal gelen meme bezlerinin iltihaplanması süt iltihaplı hücrelerin bir artış ile sonuçlanır. Bunun bir sonucu olarak, somatik hücre koSütteki unts (SCC), çoğu ülkede süt kalitesinin önemli bir göstergedir, azalan süt fiyatı ya da teslim durağına giden artar. Fournier ve ark., 2 RS-PCR yanı sıra, diğer yazma yöntemleri S. sığır suşları alt tiplerinin için tarif edilmiştir aureus 8-11. İki ortak olanları spa yazarak 12 ve Multi Odağı Sırası Yazma (MLST) 13 içerir. Ikinci bir 7 hazırlık geninin sıralama gerektirir, oysa önceki bir protein A stafilokok Spa kodlayan genin değişken aralayıcı bölgesini DNA dizilimi dayanır. Bu, bir 12 ve yedi PCR 13, sırasıyla özel ekipman kullanılarak amplikon saflaştırma, sıralama reaksiyonunun ve analiz, ardından yapılması gerektiği anlamına gelir. RS-PCR ile karşılaştırıldığında, bu yöntem, düşük seviyede örnek ve yüksek maliyetleri ile sonuçlanan laboratuarda önemli bir ek bir çaba gerektirir.verim PFGE için özellikle düşüktür. S. sığır suşları için bu yöntemlerin çözünürlüğü göz önüne alındığında aureus, RS-PCR, en azından MLST 4 veya PFGE 15 den 4 ve daha iyi yazarak spa kadar iyidir. İkili yazma 13 dahil olmak üzere tüm bu yöntemler S. neden sığır IMI patogenezinde içine daha fazla bilgi elde etkinliğini gösterdi aureus, ancak çünkü söz konusu kısıtlamalar onlar büyük klinik araştırmalar için daha az uygundur. Buna ek olarak, RS-PCR yöntemi ile genotipleme Fournier ve arkadaşları tarafından tarif edildiği gibi. 2 epidemiyolojik ve S patojenik potansiyeli güvenilir bir tahmin sağlar aureus, sığır IMI klinik veteriner hekimlikte iki anahtar değerleri çıkıyor.

<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.

Bir genotip ve türevleri tanımı belirlenen tüm genotipler birden fazla bant farklı bir elektroforetik profilini yeni genotip olarak kabul edilir. Yeni genotipleri adlandırılır ve genişletilmiş Fournier ve diğerlerine göre. 5 GTAZ genotiplerin GTAA ardından GTZ genotiplerin GTA giden ve GTBA GTBZ için ve GTCC için GTCA düzeydedir. sadece bir bant değişimin, bir genotip bir varyasyon olarak ...

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.

bir Genotiplendirme<em&gt; Staphylococcus aureus</em&gt; Tarafından Ribozomal 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, ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

9.6K Görüntüleme.  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

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.

Nesil bir gen kesintiye Streptococcus mutans çoğunlukla zorlanma olmadan Gen Klonlama

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 ...

Genetik

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

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Kan örneklerinden Bakteriyel Patojen Algılama ve Antimikrobiyal Direnç Testi için bir günlük iş akışı şeması

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

İmmünoloji ve Enfeksiyon

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.

Antibiyotik Dirençli Stafilokok bakteri tespiti için biyosensör

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

Biyomühendislik

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.

Metisiline diren

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-Benzeşim Saflaştırma gram-pozitif bakterilerde RNA Dizilimi ile birleştiğinde

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

Biyokimya

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.

16S rRNA gen Sıralama ve MALDI-TOF MS tarafından Nadir Bakteriyel patojenlerin tanımlanması

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.

Genom çapında Gen Delesyonlar içinde<em&gt; Streptococcus sanguinis</em&gt; Yüksek Verimli PCR ile

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

Biyoloji

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.

16S Ribozomal RNA Gen amplikonlarının nesil Sıralama

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.

Bakteriyel Topluluk Karakterizasyonu Verimli Nükleik Asit Ekstraksiyonu ve 16S rRNA gen Sıralama

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.

İki adımlı PCR ve Yeni Nesil 16S rRNA Gen Sıralaması Kullanarak Mikrobiyota Analizi

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

bir Genotiplendirme

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