이 작품에 대한 자금 지원은 자연 과학 및 캐나다의 공학 연구 협의회 (NSERC)과 센티넬 Bioactive 종이 네트워크에 의해 제공되었다.

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관심의 어떠한 충돌 선언 없습니다.

일반 세균의 검출 방법의 대부분은 오늘도 느려 (클래식 미생물) 또는 기술적 요구 (항체, PCR)입니다. 따라서, 우리는 탐지 도구의 차세대 속도와 단순으로 수용해야한다고 믿습니다. 이를 위해, 우리는 RNA-cleaving 및 형광 신호의 생성을 통해 박테리아의 존재를보고 간단한 assays을 개발하는 데 사용할 수있는 형광 - 신호 DNAzyme을 만들었습니다. 기능 DNAzyme 프로브, RFD-EC1은 E.의 성장 기간 동안 생산 CEM에 의해 활성화됩니다 문화 미디어의 대장균. 우리의 방법은 감지의 대상으로 세균의 원유 세포외 혼합물을 사용하며, 힘드는 대상 추출 및 증폭 단계를 무시하기 때문에, 그것은 매우 간단한 설정하는 데 사용할 수 있습니다, 세균 검출을위한 assays의 유형을 &#39;믹스 앤 읽기 &quot;. 우리 DNAzyme의 사용은 형광 기반의 검출 방법에 제한되지 않습니다. 예를 들어, colorimetric 탐지 같은 DNAzyme 시스템 분석 C를 사용은 유기 염료와 함께 압연 원형 증폭을 이용 이전에 보고된 방법을 사용하여 설계한다. 32 우리는 더 큰 운영 단순 새로운 세균 biosensors를 생성하는 매력적인 가로수와 같은 세균 검출을위한 DNAzymes의 사용을 예견한다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 또는 모델 </td></tr><tr><td> 한천 </td><td> BioShop 캐나다 </td><td> AGR003 </td></tr><tr><td> 암모늄 persulfate (APS) </td><td> BioShop 캐나다 </td><td> AMP001 </td></tr><tr><td> 아크릴 아미드 / 비스 아크릴 아미드 (40 %, 29:1) </td><td> BioShop 캐나다 </td><td> ACR004 </td></tr><tr><td> 붕산 </td><td> BioShop 캐나다 </td><td> BOR001 </td></tr><tr><td> Bromophenol 블루 </td><td> 시그마 - 올드 리치 </td><td> B8026 </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> EM 과학 </td><td> EXO539-1 </td></tr><tr><td> HCL </td><td> 시그마 - 올드 리치 </td><td> 38,281 </td></tr><tr><td> HEPES </td><td> Bioshop 캐나다 </td><td> HEP001 </td></tr><tr><td> LB의 국물 </td><td> 시그마 - 올드 리치 </td><td> L3022 </td></tr><tr><td> MgCl 2 </td><td> EMD화학 제품 </td><td> B10149-34 </td></tr><tr><td> NaCl </td><td> BioShop 캐나다 </td><td> SOD002 </td></tr><tr><td> NaOAc </td><td> EMD 화학 </td><td> SXO255-1 </td></tr><tr><td> NaOH </td><td> EMD 화학 </td><td> SXO590-1 </td></tr><tr><td> SDS </td><td> BioShop 캐나다 </td><td> SDS001 </td></tr><tr><td> TEMED </td><td> BioShop 캐나다 </td><td> TEM001 </td></tr><tr><td> 트리스베이스 </td><td> BioShop 캐나다 </td><td> BST666 </td></tr><tr><td> 십대 초반 20 </td><td> 시그마 - 올드 리치 </td><td> P9416 </td></tr><tr><td> 요소 </td><td> BioShop 캐나다 </td><td> URE001 </td></tr><tr><td> Xylenecyanol FF </td><td> 시그마 - 올드 리치 </td><td> X4126 </td></tr><tr><td> DNA 농축기 </td><td> 써모 과학 </td><td> 학자의 DNA Speed​​Vac 120 </td></tr><tr><td> Millex 필터 유닛 </td><td> Millipore </td><td> SLGP033RS </td></tr><tr><td> 젤로드 도움말 </tD&gt; <td> Diamed </td><td> TEC200EX-K </td></tr><tr><td> ImageQuant 소프트웨어 </td><td> 분자 역학 </td><td> 버전 5.0 </td></tr><tr><td> Kimwipes </td><td> 킴벌리 - 클락 전문 </td><td> 34,705 </td></tr><tr><td> 미니 Vortexer </td><td> VWR </td><td> 58816-121 </td></tr><tr><td> Parafilm </td><td> Pechiney 플라스틱 포장 </td><td> PM996 </td></tr><tr><td> 배양 접시 </td><td> 피셔 과학 </td><td> Fisherbrand 08-757-12 </td></tr><tr><td> Stripettor 플러스 (피펫 총) </td><td> 코닝 </td><td> 07764714 </td></tr><tr><td> 석영 cuvettes </td><td> Varian INC </td><td> 66-100216-00 </td></tr><tr><td> 흔드는 / 보육 </td><td> 뉴 브 런 즈윅 과학 </td><td> 클래식 시리즈 C24 </td></tr><tr><td> 태풍 스캐너 </td><td> GE 헬스케어 </td><td> 9200 가변 모드 </td></tr><tr><td> 원심 분리기 </td><td> Beckman 보습 바로 앞에 달린 풀베는 날 </td><td> Allegra X22-R </td></tr><tr><td> 자외선 분광 </td><td> 써모 과학 </td><td> GenesysUV 10 </td></tr><tr><td> 형광 분광 </td><td> Varian INC </td><td> 캐리 이클립스 </td></tr></tbody></table>

detection of bacteria using fluorogenic dnazymes

발병은 사망과 입원의 수백만뿐만 아니라 어마어마한 경제적 손실을 각각 매년 식인과 병원 - 인수 병원균 계정에 연결되어. 지속적인 유행병 장소 정확하게 초기 단계에서 범인의 병원균을 식별할 수있는 분석 방법에 대한 늘어나는 수요의 영향 등의 발생과 최소화의 예방. 높은 특이성 (이익만을 세균 검출), 고감도 (낮은 검출 능력 : 병원균 검출을위한 효과적인 방법의 큰 배열이있다지만, 그들 모두가 이상적인 검출 방법에 대한 구현 모두 다음과 같은 다섯 최고의 요건을 충족 없습니다 하나의 살아있는 세균 세포 등), 짧은 시간 - 결과 (시간 분), 훌륭한 운영 단순 (긴 샘플링 절차 및 전문 장비의 사용에 대한 필요도), 그리고 비용 효율성. 예를 들어, 고전적인 미생물 방법은 매우 구체적인 것이지만 오랜 시간 (다를 필요로주 YS는) 1 PCR과 항체 기반 기술을 짧은 대기 시간 (일 시간) 제공합니다. 최종 결과를 얻기 위해,하지만 그들은 값비싼 시약 및 / 또는 정교한 장비의 사용. 따라서 2-4 요구, 아직 거기있다 상기 요건 중 하나 이상의 향상된 특징을 제공하는 혁신적인 세균의 검출 방법을 개발하는 방향으로 과학 연구에 대한 큰 수요. 우리 연구실은 세균 검출 포함한 biosensing 어플 리케이션을위한 분자 프로브의 소설 클래스로 DNAzymes의 가능성을 검사에 관심있다 5. DNAzymes는 (또한 deoxyribozymes 또는 DNA의 효소라고도 함)가 사람이 만든 화학 반응을 catalyzing의 기능이 단일 좌초된 DNA의 분자를. 6-8 이들 분자가 많은 10대로 들어있는 광대한 임의 시퀀스의 DNA 풀 (격리 수 과정에 의한 16 개별 시퀀스)가 O &quot;체외 선택에서&quot;로 알려진R &quot;SELEX&quot;(지수 농축에 의한 리간드의 체계적 진화). 9-16 이러한 특수한 DNA 분자 널리 biosensing 응용 프로그램을위한 분자 도구로 최근 몇 년 동안 검사되었습니다. 6-8 우리 연구실은 RNA-cleaving 형광 DNAzymes (. RFDs; 그림 1)을 분리을위한 체외 선택 절차에 ​​설립되었으며 분석 도구로 RFDs의 사용을 조사 17-29 RFDs가 단일 ribonucleotide에서 DNA-RNA 키메라 기판의 절단을 catalyze. 형광단 (F)과 끄는 (Q)의 어귀되어 접합 (R). F와 Q의 가까운 거리는 uncleaved 기판 최소한의 형광을 렌더링. 그러나 절단 이벤트는 형광 강도의 상당한 증가와 함께되어 F와 Q의 분리로 안내합니다. 최근, 우리는 박테리아 감지를위한 RFDs를 분리하는 방법을 개발 5.이 특별한 RFDs은 &quot;으로 고립되었다자신의 환경이나 그들이 배양해되는 미디어 박테리아의 특정 유형 (그림 1)에 의해 남겨진 원유 세포외 혼합물 (CEM)의 면전에서 &quot;점등. 원유 혼합물의 사용이 정화의 지루한 과정을 circumvents 및 바이오 센서 개발 (완료하는 데 몇 달 또는 몇 년이 걸릴 수도있는)에 대한 관심 미생물의 적합한 대상을 식별. 세포외 대상의 사용은 세포내 표적을 구하는 단계 필요가 없기 때문에 시금 절차는 간단하다 의미합니다. 오직 E. 제작한 CEM의 면전에서, 위의 접근 방식을 사용하여, 우리는 (그림 2A. FS1)은 기판 클리브 RFD를 파생 대장균 (CEM-EC) 5.이 E. RFD-EC1 (그림 2A)이라는 대장균 감지 RFD는, 엄격하게 CEM-EC에 대응하지만 다른 박테리아의 호스트 (그림 3)에서 CEMs에 대한 의식이된다는 것을 발견했다. 여기 preseE.을 설정하기위한 주요 실험 절차를 NT RFD-EC1 및 담당자 결과를 이용하여 대장균 검출 assays.

Watch this Scientific Journal Video about Detection of Bacteria Using Fluorogenic DNAzymes at JoVE.com

Detection of Bacteria Using Fluorogenic DNAzymes

Google은 최근은 간단 세균 검출을위한 형광 분석 &quot;을 믹스 앤 읽기&quot;를 설정하는 데 적용할 수있는 형광 DNAzyme 프로브를 생성하기위한 새로운 접근 방식을보고했습니다. 이러한 특별한 유전자 프로브는이를 형광 신호 생성에 세균 검출을 번역 특정 세균에 의해 생산되는 원유 세포외 혼합물 (CEM)의 앞에서 발색단 - 수정의 DNA-RNA 키메라 기판의 절단을 catalyze. 이 보고서에서 우리는 &quot;RFD-EC1&quot;표시된 특정 DNAzyme 프로브는 모델 박테리아의 탐지를 위해 고용되는 핵심 실험 절차를 설명합니다<em&gt; 대장균 (E. 대장균)</em&gt;.

형광 DNAzymes을 사용하여 박테리아의 탐지

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

detection-of-bacteria-using-fluorogenic-dnazymes

Research

JoVE Journal

Immunology and Infection

18.8K 조회수.  McMaster University. Google은 최근은 간단 세균 검출을위한 형광 분석 "을 믹스 앤 읽기"를 설정하는 데 적용할 수있는 형광 DNAzyme 프로브를 생성하기위한 새로운 접근 방식을보고했습니다. 이러한 특별한 유전자 프로브는이를 형광 신호 생성에 세균 검출을 번역 특정 세균에 의해 생산되는 원유 세포외 혼합물 (CEM)의 앞에서 발색단 - 수정의 DNA-RNA 키메라 기판의 절단을 catal...

비디오: Detection of Bacteria Using Fluorogenic DNAzymes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

접착제 테이프 기반의 샘플링 및 형광의 조합 현장에서</em의 신속한 감지를 위해> 하이브 리다이 제이션 살모넬라균 신선한 농산물에 대한

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

연구

면역학 및 감염병학

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Oligonucleotide - 결합 형광등 Microspheres를 사용하여 복잡한 임상 및 환경 샘플에서 박테리아의 멀티 플렉스 감지

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF 질량 분석기를 사용하여 혈액 배양액에서 그람 음성 박테리아의 빠른 확인

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

16S rRNA의 타겟팅 디곡신 표지 DNA 프로브를 사용하여 파라핀 조직 내에서 박테리아의 제자리 검출

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

분자 각 인을 사용 하 여 생체의 磁 검출 용량 성 바이오 센서 기반

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

살 모 넬 라 종의 심사에 대 한 높은 처리 플랫폼 /Shigella 종

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

무기 Polyphosphate 박테리아에 대 한 시 금

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

유세포측정을 사용하여 공생 균에 결합하는 인간 노로바이러스 바이러스와 같은 입자 정량화

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

혐기성 박테리아에 의한 쥐 질 집락화 모델

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

Fluorescent Reporter 정량화를 통한 전체 장기 병원체 추적

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

저자 스포트라이트: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues(조직의 항생제 및 면역 체계에 대한 박테리아 반응 규명)

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

고분해능 3차원 전장기 단층촬영(High-resolution three-dimensional whole-organ tomography of microbial infections)

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...