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. 황색 포도상 구균 분리 된 균주 <ol><li> S.의 10 μl를 확산 구균 재고 문화 또는 하나의 식민지가 5 % 양 혈액 (BA)를 포함 컬럼비아 아가 플레이트에 선택 배지에 성장. </li><li> 24 시간에 18 시간 동안 37 ° C에서 기적 품어. </li></ol> 2. DNA 추출 <ol><li> 10 mM 트리스 - 염산 10 밀리미터 나 2 EDTA, pH가 8.5를 포함 TEL 버퍼를 준비합니다. 15 분 동안 121 ° C에서 분취 및 오토 클레이브를 준비합니다. </li><li> 100 μl의 전화에서 멸균 플라스틱 루프 또는 멸균 나무 이쑤시개와에 resuspend를 사용하여 BA 1 식민지를 수집합니다. 스크류 캡 1.5 ml의 튜브를 사용합니다. </li><li> 10 분 동안 95 ° C에서 품어. 그 후, 젖은 얼음에 즉시 샘플을 배치합니다. </li><li> PCR (PCR 용 = 주형 DNA)에 적합한 H 2 O 100 : 샘플 1을 희석. 비 희석하고, 희석 된 샘플을 1 달 동안 -20 ℃에서 보관 될 수있다. </li></ol> 3. RS-PCR <ol><lPCR의 튜브 1.5 μL의 H에 각각의 샘플 피펫 용 I&gt; 2 O, DNA 형성 촉매 마스터 믹스 μL 12.5, 2 μL의 G1 프라이머 10 μM, 2 μL L1 프라이머 10 μM, 순수의 약 30 NG에 해당하는 주형 DNA의 7 μL DNA 2. </li><li> 표준 PCR의 자전거 타는 사람에서 다음 사이클 프로그램을 실행 : 15 분 7 분 55 ° C에서 2 분 램프 및 어닐링 다음에 1 분 94 ° C를 포함하는 27주기, 다음 95 ° C는. 추가의 2 분의 램프 후, 2 분 동안 72 ℃에서 연장된다. 4 ℃로 냉각 한 다음 10 분 동안 72 ° C에서 최종 연장 PCR을 종료합니다. 주 : PCR 산물을 5 일 동안 -20 ℃에서 저장 될 수있다. </li><li> 각 실행에 대한 RS-PCR, 즉 양성 대조군, 공지 된 유전자형 (일반적 GTB)와 샘플 및 대조군을 포함 (H 2 O). </li></ol> 4. 전기 영동 <ol><li> 함께 DNA (MED 키트)에 대한 상용 소형 전기 키트를 사용하여PC와 설치된 소프트웨어에 연결된 bioanalyzer와. 조심스럽게 제조 업체의 대응 매뉴얼을 공부한다. </li><li> 칩 프라이밍 스테이션을 설정하고 제조업체의 설명서에 따라 주사기 클립을 조정합니다. </li><li> 제조업체의 설명서에 따라 젤 염색 믹스를 준비합니다. </li><li> (제조자의 설명서에 따라) 젤 염색 믹스로드. <ol><li> 사용하기 전에 30 분 동안 실온으로 겔 염료 혼합물 평형. </li><li> 칩 프라이밍 스테이션에서 새로운 DNA 칩을 넣습니다. 잘 표시 G.에서 젤 염료의 피펫 9 μl를 </li><li> 플런저 1 ml의에 위치되어 있는지 확인하고 칩 마중물 역을 닫습니다. 를 눌러 플런저은이 주사기 클립에 의해 개최 될 때까지. 30 초 후 주사기 클립을 해제에 대해 정확히 기다립니다. </li><li> 5 초 동안 기다립니다. 천천히 1 ml의 위치로 플런저를 다시 잡아 당깁니다. </li><li> 두 우물에서 열기 칩 프라이밍 스테이션과 젤 염료의 피펫 9 μl의는 &#39;G&#39;를 표시했다. </li></ol></li><li> 는 M로드arker <ol><li> 모든 12 샘플 우물에 잘 사다리에있는 마커의 피펫 5 μL. 빈이 13 우물 중 하나를 두지 마십시오. </li></ol></li><li> 래더 및 샘플을로드. <ol><li> 피펫 래더 기호 잘 표시에 DNA 사다리 1 μL. </li><li> 샘플의 피펫 1 μl를 (사용 우물) 또는 탈 이온수 (사용하지 않는 우물) 1 μL. </li><li> 2,400 rpm에서 1 분간 어댑터와 소용돌이에 수평으로 칩을 넣습니다. </li><li> 5 분 이내에 bioanalyzer에서 칩을 실행합니다. </li></ol></li><li> 분석을 실행 <ol><li> bioanalyzer와 연결된 컴퓨터를 시작합니다. 소프트웨어를 엽니 다. </li><li> bioanalyzer의 뚜껑을 열고 (칩이 한 방향으로 만 맞는) 칩 홀더에 칩을 놓습니다. 조심스럽게 뚜껑을 닫습니다. </li><li> 소프트웨어의 악기 맥락에서 &quot;전기 영동을&quot;&gt; &quot;dsDNA&quot;&gt; &quot;DNA 7500&quot;을 선택합니다. 소프트웨어의 현재 파일 접두사를 그대로 사용하거나 수정합니다.</li><li> 소프트웨어에있는 시작 버튼을 클릭합니다. 소프트웨어에서 샘플 이름을 입력합니다. 칩 실행이 완료되면 (후 약 30 분) 실행 메시지의 끝은 bioanalyzer 소프트웨어에 나타납니다. </li><li> 제조자의 지시에 따라 bioanalyzer의 전극과 상기 칩을 제거하고 세정. </li></ol></li></ol> 5. 전기 영동 프로필에서 유전자형을 유추 <ol><li> (소프트웨어는 저자에서 자유롭게 사용할 수 있습니다)을 마 소프트웨어를로드합니다. </li><li> &gt; &quot;가져 오기 참조 데이터&quot;, &quot;파일&quot;다음 마할 소프트웨어로 가져 오기 참조 데이터 </li><li> bioanalyzer 소프트웨어의 데이터 컨텍스트에서 실행되는 전기를 엽니 다. 하나의 샘플의 전기 영동 프로파일을 선택합니다. </li><li> 관련 피크를 확인합니다. <ol><li> &#39;도구&#39;&gt; &#39;평균 형광 &quot;관련 피크를 확인하기 위해 마 소프트웨어를 사용합니다. 은 &quot;형광&quot;상자에 삽입형광 값은 그들이 bioanalyzer 소프트웨어로부터 판독 될 수있는 형광 단위 (FU)로 나타내었다. 오름차순으로 가장 높은 형광 4 (3) 피크를 삽입하고 쉼표 (,)로 구분. 예 : 126,130,132,137. 참고 : 모든 이들 피크의 FU의이 bioanalyzer 소프트웨어로 표시하는 경우, 그들은 음모에서 그들을 판독하여 추정 할 필요가있다. </li></ol></li><li> 은 &quot;계산&quot;버튼을 누릅니다. 관찰 된 형광은 박스 &quot;최소 피크 높이&quot;에 소정의 값을 초과하면, 피크가 적합하다. </li><li> 유전자형을 추론. <ol><li> 마할 텍스트 상자 &quot;입력 봉우리 (BP)&quot;를 오름차순으로 모든 관련 피크의 염기쌍의 크기와 쉼표 (,)로 구분으로 삽입합니다. 예 : 440,462,519,579,637. 주 : 이러한 크기의 피크는 모든 bioanalyzer 소프트웨어에 의해 지시되는 경우에, 플롯 그들을 판독하여 추정 될 필요가있다. </li></ol></li><li> 은 &quot;계산&quot;버튼을 누릅니다. </li><li> 목록에서상자, 최소 제곱 마할 라 노비스 거리 &quot;D2M&quot;를 찾습니다. 이 ≥0.05 인 오른쪽에 대응하는 P 값은 그 확률 귀무 가설 H 0이 거부되었는지 ≥5 % 인 경우 (H 0 : 관찰 기준 프로파일이 동일) 관찰 된 프로파일과 동일하도록 허용되는 것을 의미 지정된 유전자형의 기준 프로파일. </li><li> D2M 최소화하지만 해당 P 값의 필드가 비어 있으면, 확률 &lt;5 % H 0을 거부한다. 참고 :이 D2M 최소한 있지만 프로파일이 동일하지 않은 것을 의미합니다. 이러한 결과는 비 평균 전기 영동 조건에서 발생할 수 있습니다. <ol><li> 이러한 편차를 보정하려면 값 395을 &quot;피크 (BP) 관찰&quot;텍스트 상자에 삽입하고 &quot;계산&quot;버튼을 누릅니다. 최소한의 D2M와 대응하는 P 값 ≥0.05 다시 확인합니다. 성공하지 않으면, 값 405, 415 및 425을보십시오. 참고 : 때때로 작은 단계도 있습니다가리키는. 관찰 된 프로파일이 기준 데이터가 존재하지 여전히 성공적이지 않으면 새로운 유전자형 또는 변형 될 수있다. </li></ol></li><li> 다양한 프로필 <ol><li> 반복 별도로 5.6 각 전기 프로파일 5.9에 단계를 반복합니다. </li></ol></li></ol>

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The authors have nothing to disclose.

RS-PCR (&gt; 30 순수한 DNA / 분석 겨)에서 DNA의 초과가있을 때 전체 공정의 가장 중요한 단계는이. 그 피크가 모두 기준선에서 시작 및 종료하는 경우 일반적으로, 프로파일의 해상도가 최적이다. 두 개의 피크가 서로 너무 가까이있는 경우, 해상도는 완전하지 않습니다. 수동 식별을 필요되도록 이러한 조건 bioanalyzer 소프트웨어 부적절한 피크 식별을 초래할 수있다. BP ?...

황색 포도상 구균 (S. 구균) 소 1 유방 내 감염 (IMI)에 대한 전 세계적 책임을 가장 중요한 병원체로 알려져있다. 12 유럽 국가 그 소에 Cosandey 등. 3, 보스 등. (4)에 의해 푸르니에 등. 2 스위스 암소에서 설명하고 연구의 연구 S. 구균은 IMI는 유 전적으로 이질적인 그룹 소에서입니다. 16S-23S rRNA의 유전자 간 스페이서 영역 (RS-PCR)의 PCR 증폭에 의해, 17 유전자형 총 초기 101 역학적 분리 독립이 검출되었다. 다른 15 유전자형 GTS (가) 각 1 모든 균주의 % 4을 거의 발생하지 반면 유전자형 B 및 유전자형 C (GTC)는 (80 %) 가장 많았다. 유럽 레벨 3에서 GTB는 GTC는 GTF, GTI, 그리고 GTR은 분석 된 모든 균주 (N = 456)의 76 %를 포함하는 가장 중요한 유전자형이었다. GTB는 w 중부 유럽에 위치하고했다hereas 다른 유전자형 널리 전파되었다. 균주의 나머지 24 %는 그들 중 많은,하지만, 한 번만 관찰되었다 (41) 유전자형을 포함. 유전자형이 높은 유럽 수준으로 스위스에서뿐만 아니라, 그들의 독성 유전자 패턴과 연관되었는지 포니 외. 2 Cosandey 외. (3), Graber 외. (5)에 의한 연구는 또한 보여 주었다. 연구는 또한 S. 중 IMI 보급에 엄청난 차이를 밝혀 구균 GTB, GTC 다른 유전자형 2,5 : GTB 고려, 무리 암소의 87 %까지이 유전자형에 감염되었다. GTC의 다른 유전자형의 경우에, 그러나, IMI은 무리 1-2 소에서 발견되었다. IMI S.에 의해 발생 구균은 일반적으로 해당 유방 동맥의 염증과 우유에서 염증 세포의 증가를 초래한다. 결과적으로, 체세포 CO우유에 unts (SCC), 대부분의 국가에서 우유 품질의 주요 지표는 감소 우유 가격 또는 납품 정지로 이어지는 증가한다. 포니 외. (2)의 RS-PCR 외에, 다른 입력 방법은 S.의 소 하위 유형 균주에 대해 기재 한 구균 8-11. 두 가지 일반적인 것들로는 스파 입력 (12)과 멀티 자취 그리기 순서 타이핑 (MLST) (13)를 포함한다. 후자 7 하우스 키핑 유전자의 염기 서열을 필요로하는 반면, 전 하나, 단백질 A에 대한 포도상 구균 스파 코딩하는 유전자의 가변 영역을 스페이서 DNA 시퀀싱에 기초한다. 이것은 하나의 12 일곱 PCR들 (13) 각각의 특정 장치를 사용하여 앰플 리콘 정화 시퀀싱 반응을 분석 한 후, 수행 될 필요가 있음을 의미한다. RS-PCR에 비해, 이러한 방법은 낮은 샘플 처리량과 높은 비용의 결과로 실험실에서 상당한 추가적인 노력이 필요합니다. 그만큼처리량은 PFGE 특히 낮습니다. S.의 소 균주에 대한 이러한 방법의 해상도를 고려 구균, RS-PCR 적어도 MLST 4 PFGE (15)보다 4 더 나은를 입력 스파만큼 좋은 것입니다. 바이너리 입력 (13)을 포함한 모든 이들 방법은 S.으로 인한 소 IMI의 발병에 더 통찰력을 얻기에 그 효과를 입증 구균,하지만 때문에 언급 제한으로 그들은 많은 임상 연구 덜 적합하다. 또한, RS-PCR에 의한 유전자형 포니 등에 의해 설명 된 바와 같이. 2 역학적 S.의 발병 가능성을 예측 신뢰성있게 구균, 소 IMI 임상 수의학에 두 개의 키 값을 포함했다.

<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.

유전자형과 그 변종의 정의 식별 된 모든 유전자형에서 하나 이상의 주파수 대역이 상이한 전기 영동 프로파일은 새로운 유전자형으로 간주된다. 새로운 유전자형은 이름과 확장 푸르니에 등에 따라. 5 GTAZ의 유전자형 GTAA 다음 GTZ의 유전자형 GTA, 선도 및 GTBA GTBZ, 그리고 GTCC에 GTCA 현재된다. 한 밴드의 변화는 ?...

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.

의 유전자형<em&gt; 황색 포도상 구균</em&gt;에 의해 리보솜 스페이서 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 조회수.  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.

비디오: 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.

유전자 교란 mutans 연쇄 상 구 균 긴장 없이 유전자 복제의 생성

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 ...

연구

유전학

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.

혈액 배양에서 세균 병원체 탐지 및 항균 저항 테스트를위한 하루 워크플로 계획

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

면역학 및 감염병학

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.

항생제 내성 황색 포도상 구균 박테리아의 검출을위한 바이오 센서

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

생체공학

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.

Methicillin - ??? V? ??? ?? ??? ??? ? ?? I? ?????? ?? ??? PCR ?? ??<em&gt; ?? ??? ??</em

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;

그람 양성 박테리아에서 RNA 염기서열분석과 결합된 MS2-선호도 정화

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

생화학

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 rRNA의 유전자 시퀀싱과 MALDI-TOF MS에 의해 희귀 세균성 병원체의 식별

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.

게놈 전체 유전자 삭제할에<em&gt; 연쇄상 구균 sanguinis</em&gt; 높은 처리량 PCR에 의한

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

생물학

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 리보솜 RNA 유전자 증폭 산물의 차세대 시퀀싱

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.

세균 커뮤니티 특성화에 대한 효율적인 핵산 추출 및 16S rRNA의 유전자 시퀀싱

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.

2단계 PCR 및 차세대 16S rRNA 유전자 시퀀싱을 사용한 미생물 분석

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

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