이 연구는 해군 의료 연구 센터, 연구 작업 단위 6000.RAD1.J.A0310에 의해 지원되었다. 이 문서에 표현 된 의견은 저자의 것이며, 반드시 해군, 국방부, 또는 미국 정부의 부서의 공식적인 정책이나 입장을 반영하지 않습니다. 웨이 메이 칭은 미국 정부의 직원이다. 이 작품은 그녀의 공무의 일환으로 제조 하였다. 제목 17 USC §105은 &#39;이 제목에서 저작권 보호는 미국 정부의 모든 작업에 사용할 수 없습니다.&#39;고 제공 제목 17 USC §101은 그 사람의 공무의 일환으로 병역 구성원 또는 미국 정부의 직원에 의해 제조 된 작품으로 미국 정부의 작업을 정의합니다.

1. LAMP 반응에 대한 표준으로 플라스미드 DNA 희석 준비 <ol><li> DNA 농도 플라스미드 (C. burnetii RSA 493의 포함 대상 유전자 IS1111a)를 결정하기 위해 260 nm에서 광학 밀도 (OD)를 측정하는 분광 광도계를 사용합니다. (1)의 OD (260)는 DNA의 50 μg의 / μL 동일합니다. </li><li> 카피 수에 플라스미드 DNA의 양을 변환합니다. 플라스미드의 크기는 6,330 bp의이기 때문에,이 플라스미드의 분자량은 4.1 × 106 G (6330 BP X 649g / BP)이다. (단계 1.1 결정) 플라스미드 DNA의 농도는 34.2 NG / μL이다. 이 DNA 샘플 1 μL는 DNA의 5 × 10 9 사본 (34.2 × 10 -9 g / 4.1 × 10 6 GX 6.02 × 10 23)에 동일시, 플라스미드 DNA의 34.2 NG가 포함되어 있습니다. </li><li> 5 × 8 사본 DNA 샘플을 만들기 위해 10 배 희석 물을 90 μL로 플라스미드 DNA (5 × 109 카피 / μL)의 10 μL를 추가 / μL. </li><li> 반복 단계 1.3, 5 × 7의 DNA 샘플을 준비하기 위해 5 × 106, 5 × 105, 5 × 104, 5 × 103, 5 × 2, 5 × 10 1 5 X 10 0 사본 / μL. </li></ol> 2. LAMP 반응에 대한 샘플에서 DNA 템플릿을 준비 <ol><li> 제조사의 프로토콜에 따라 상업적 DNA 미니 키트를 사용하여 인간 혈장 시료로부터 DNA 추출을 수행한다. 추출에 200 μL 샘플의 전체를 사용하여 20 ㎕의 부피에서 추출한 DNA를 용출시킨다. </li></ol> 3. 배 LAMP 반응 버퍼를 준비합니다 <ol><li> 10 배 폴 버퍼 200 μL, 5 M 트리메틸 320 μL, M 황산 4,의 dNTP 혼합물의 280 μL (10 mM의 각) 및 물 188 ㎕의 2 배 LAMP 버퍼의 1 mL로 한 12 μl를 섞는다. 10 초 동안 펄스 텍싱에 의해 섞는다. </li></ol> 4. 표준 LAMP 반응을 수행 <ol><li> 배 LA의 12.5 μl를 혼합3 단계에서 제조 된 MP 완충액 (40 mM 트리스 - 염산 (PH 8.8), 20 mM의 KCl을 16 mM의 황산, 20 mM의 (NH4) 2SO4, 0.2 % 트리톤 X-100, 1.6 M 트리메틸 1.4 밀리미터의 dNTP 혼합물)의 프라이머 혼합물 1.2 μL, BST DNA 중합 효소 (8 U / μL) 1 μL, 및 0.2 ㎖의 PCR 용 튜브에 반응 혼합물 (20 μL)을 만들어 물 5.3 μL. </li><li> 20 ㎕의 반응 혼합물에 5 (1 단계 또는 2) DNA의 μl를 넣고 잘 섞는다. </li><li> 근접 튜브는 수조 또는 히팅 블록에 60 분 동안 60 ℃에서 배양한다. 이는 앞서 결정된 최적의 반응 온도이다. </li><li> 반응을 종료 10 배 겔 로딩 버퍼의 5 μl를 추가합니다. </li><li> 핵산 얼룩 인터 물들 2 % 아가 로스 젤에 반응 생성물의로드 5 μL. </li><li> 1X TBE 버퍼에서 35 분 동안 100 V에서 젤을 실행합니다. </li><li> 자외선에 의한 결과를 시각화합니다. </li></ol> 5. 재구성와 LAMP 반응을 수행시약 <ol><li> 10 배 폴 버퍼 125 μL, 5 M 트리메틸 200 μL, 1 M 황산의 7.5 μl를 혼합, 물 667.5 μL를 재구성 버퍼의 1 mL로한다. 10 초 동안 펄스 텍싱에 의해 섞는다. </li><li> 밀봉 된 알루미늄 호일 가방에서 동결 건조 된 시약을 포함하는 튜브를 제거합니다. 알루미늄 박 백이 밀폐되지 않으면 튜브를 사용하지 않는되거나 손상된 경우. </li><li> 동결 건조 된 시약을 다시 중단 각각의 튜브에 재구성 버퍼의 20 μl를 추가합니다. </li><li> 버퍼 위아래로 5 회 피펫 팅하여 섞는다. 동결 건조 된 시약을 완전히 재현 탁하고 있는지 확인하기 위해 살펴 보자. 백색 분말은 튜브에 사라집니다. </li><li> 재구성 된 시약 (1 단계 또는 2에서) DNA 템플릿의 5 μl를 넣고 잘 섞는다. </li><li> 근접 튜브는 수조 또는 히팅 블록에 60 분 동안 60 ℃에서 배양한다. </li><li> 반응을 종료 10 배 겔 로딩 버퍼의 5 μl를 추가합니다. </li><li> 로드 반응의 5 μL핵산 얼룩 인터 물들 2 % 아가 로스 젤 제품. </li><li> 1X TBE 버퍼에서 35 분 동안 100 V에서 젤을 실행합니다. </li><li> 자외선에 의한 결과를 시각화합니다. </li></ol> 6.에서 재구성 버퍼 HNB 또는 형광 인터 염료 함유으로 LAMP 반응을 수행 <ol><li> 10 배 폴 버퍼 125 μL, 5 M 트리메틸 200 μL, 1 M의 7.5 μl의 황산, 20 mM의 HNB (또는 100 배 형광 인터 염료의 12.5 μL) 660 μL (또는 655 μL)의 물에 7.5 μl를 혼합 재구성 버퍼 1 ㎖를 확인합니다. 10 초 동안 펄스 텍싱에 의해 섞는다. </li><li> 5.6 - 단계 5.2를 반복 섹션 6에서 제조 재구성 된 버퍼를 사용합니다. </li><li> 육안에 의한 결과 (HNB 함유 반응) 또는 UV 등 (인터 형광 염료를 함유하는 반응)을 시각화. </li></ol> 7. 튜브 스캐너와 실시간 LAMP 반응을 수행 <ol><li> reconstitut에 DNA μL 5 추가형광 인터 염료, 가까운 튜브를 포함하는 튜브 스캐너에 삽입 에드 시약. </li><li> 60 ° C에서 설정 배양 온도. 60 분 동안 520 nm에서의 형광을 측정한다. </li></ol>

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저자는 공개 아무것도 없어.

이전에, 우리는 삽입 요소 IS1111a (14) 대상으로 민감하고 특정 LAMP 분석을 개발했다. 그것은 매우 C.의 다른 균주 사이에서 보존되어 있기 때문에 IS1111 소자 선택한 burnetii와 박테리아의 사본의 높은 번호 (110-7) (클레 2006). 우리의 결과는 LAMP가 Coxiella DNA의 적은 하나 염색체 사본에 상관 관계가있다 IS1111 요소의 약 25 복사본을 감지 할 수있는 것으로 ?...

작은 그람 음성 박테리아 Coxiella burnetii는 전 세계적으로 인수 공통 전염병 인 Q 열병의 원인 물질이다. 때문에 Q 열병의 전세계 유통 및 C의 높은 감염에 burnetii는 해외 배치 미군과 민간인은 발병 지역에서 감염 될 위험이 있습니다. 급성 및 만성 감염 : Q 열병은 두 가지 형태로 나타난다. Q-급성 발열 독감 유사 증상, 간염, 폐렴 자체를 제공하고, 일반적으로 낮은 사망률 자기 한정 질환이다. 만성 Q-열이 덜 유행하면서, 자주 1,2 방치하면 훨씬 더 높은 사망률을 가지고 심내막염, 발생합니다. 따라서 조기 진단 적절한 치료가 환자 치료에 중요 안내합니다. 중합 효소 연쇄 반응 (PCR) 및 실시간 정량 PCR (qPCR에) 분석법 C. 검출을 위해 개발되어왔다 세포 배양 및 임상 샘플 3-5 &lt;에 burnetii DNA/ SUP&gt;. PCR 및 qPCR에 모두 비용과 일상적인 작업을위한 자원이 제한된 지역에서 자주 쉽게 사용할 수 없습니다. 원래 Notomi 6 설명, 루프 - 매개 등온 증폭 (LAMP)는 다른 DNA 증폭 방법을 제공합니다. LAMP은 표적 유전자의 적어도 6 독립 영역을 인식 특별히 설계된 프라이머 세트와 함께 나선 치환 DNA 합성 BST DNA 중합 효소를 사용한다. LAMP의 가장 중요한 장점은 증폭 등온 조건에서 발생한다는 것이다. 따라서, 단지 수욕 가열 블록 또는 인큐베이터가 필요하다. 이 방법은 여러 리케차 7-9 병원균을 검출하기 위해 사용되어왔다. 겔 전기 영동에 의해 증폭 된 DNA 제품의 시각화에 의한 비특이적 증폭 오탐 (false positive)에서 진정한 양성을 차별화 할 수있는 가장 정확한 방법입니다. 그러나 겔 전기 영동에 관련된 절차 리소스 제한 AR위한 실용적이지 않다EAS. 몇몇 다른 방법이 반응 생성물을 검출하기 위해 개발되었다. 피로 인산 마그네슘을 형성 (10) 또는 인터 형광 염료를 사용하여 유도 된 탁도 11,12 UV 광 하에서 가시화 될 수 등이 대안은 겔을 실행하는 것보다 더 유리하다. Q 열병이 발병 13 곳 열대 및 아열대 국가에서 주위 온도가있는, 25 ° C, 37 ° C에서 보관하면 램프 시약은 1 개월 안정적이었다. 램프와 분석은 C.의 존재를 검출하기 위해 실험실에서 개발 된 플라즈마 샘플 14 burnetii. 여기서 우리는 동결 건조 된 시약의 LAMP 반응 혼합물의 제조를위한 간단한 프로토콜을 설명한다. 실온에서 저장 될 때 동결 건조 된 시약은 1 개월 안정하다. 쉽게 시각화 방법과 결합 될 때, 이는 C. 검출에 사용하는 이상적인 방법은 자원 제한 설정에서 burnetii.

<table><tbody><tr><td>LAMP primers</td><td>Eurofins MWG operon</td><td>10 nmol, salt free</td><td>Sequence designed by customer</td></tr><tr><td>Bst DNA polymerase</td><td>New England Biolabs</td><td>M0275L</td><td>8 units/ml</td></tr><tr><td>10x Thermo pol buffer</td><td>New England Biolabs</td><td>B9004S</td><td></td></tr><tr><td>dNTP mixture</td><td>New England Biolabs</td><td>N0447L</td><td>10 mM each</td></tr><tr><td>Betaine</td><td>Sigma-Aldrich</td><td>B0300</td><td>5 M, Trimethylglycine</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M3409-1ML</td><td>1 M</td></tr><tr><td>10x Bluejuice</td><td>Invitrogen</td><td>10816-015</td><td>gel loading buffer</td></tr><tr><td>SYBR green</td><td>Invitrogen</td><td>S7585</td><td>10,000x, visualize products in tubes</td></tr><tr><td>GelRed</td><td>Phenix Research Products</td><td>RGB-4103</td><td>10,000x, visualize products in gels</td></tr><tr><td>Lyophilized reagents</td><td>Gene Reach</td><td></td><td></td></tr><tr><td>Hydroxynaphthol blue</td><td>Fluka</td><td>33936-10G</td><td></td></tr><tr><td>ESEQuant tube scanner</td><td>Qiagen</td><td></td><td>real-time detection</td></tr></tbody></table>

the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii

Coxiella의 burnetii는 Q 열병을 일으키는 원인이되는 에이전트는 의무적 세포 내 세균이다. PCR 기초 진단 분석법 C. 검출을 위해 개발되어왔다 세포 배양 및 임상 시료에서 burnetii DNA. PCR은 특수 장비와 다양한 최종 사용자 교육을 필요로하며, 따라서, 특히 자원이 제한된 지역에서 일상적인 작업에 적합하지 않습니다. 우리는 C.의 존재를 검출하는, 루프 - 매개 등온 증폭 (LAMP) 분석법을 개발 환자 샘플에서 burnetii. 이 메소드는 수조 또는 히팅 블록에 60 ° C의 주위에 하나의 온도에서 수행된다. 이 LAMP 분석의 감도는 반응 당 25 카피의 검출 한계 PCR 매우 유사하다. 이 보고서는 동결 건조 된 시약 및 hydroxynaphthol 청색 (HNB) 또는 반응 인터 형광 염료와 UV 램프를 사용하여 결과를 시각화하여 반응의 제조를 설명한다. 램프 시약은 lyophili했다평탄화 및 검출 감도의 손실없이 한달 동안 실온 (RT)에 저장된다. 반응 혼합물을 준비 복잡한 단계를 포함하지 않기 때문에이 LAMP 분석법이 특히 강하다. 이 방법은 Q 열병이 발병 자원 제한 설정에서 사용하기에 이상적이다.

0.2 ML 튜브 내부의 동결 건조 램프 시약은 BST DNA 중합 효소, 프라이머, 및 dNTPs를 포함되어 있습니다. 재구성 버퍼의 20 μl를 다시 일시 동결 건조 된 시약에 사용된다. (1)가 새로 제조 한 시약 및 동결 건조 시약 아가 로스 젤에 LAMP 반응 결과를 보여줍니다. 동결 건조 된 시약의 활성을 감소하지 않습니다. DNA 주형.도 2의 25 카피를...

Watch this Scientific Journal Video about The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii at JoVE.com

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

의 탐지를위한 동결 건조 루프 - 매개 등온 증폭 시약의 개발<em&gt; Coxiella burnetii의</em

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

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Research

JoVE Journal

Immunology and Infection

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

10.5K 조회수.  Naval Medical Research Center. We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

비디오: The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

루프 매개 이소르말 증폭을 사용하여 관개물에서 피토프토라 캡시 검출

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

연구

환경과학

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

면역학 및 감염병학

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect&#160;T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

트리파노소마 크루지 또는 기타 병원성 유기체의 DNA 검출을 위한 즉시 사용 가능한 qPCR

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a ...

생화학

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

적용 형광 공명 에너지 전달 (FRET)을하여 이펙터 전좌 효율을 검사합니다<em&gt; Coxiella burnetii의</em&gt; siRNA를 음소거시

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

모 세관 배열에 여러 개의 핵 산의 비주얼 검색

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

생체공학

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification.

자원 제한 환경에서 실시간 이소레말 핵산 증폭 반응을 모니터링하는 오픈 소스 소형 플루오리미터

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with ...

등온 증폭을 이용한 인간 호흡기 세균 감염 진단

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

저자 스포트라이트: Advancing Rapid detection of respiratory Pathogens Using Microfluidic Chip(미세유체 칩을 사용한 호흡기 병원체의 신속한 검출 발전) 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Microfluidic-Chip-based Loop-Mediated Isothermal Amplification을 통해 하기도 감염을 일으키는 세균성 병원체의 신속한 검출

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

동물성 식품에서 살모넬라를 선별하고 문화 격리에서 살모넬라를 확인하기 위한 루프 중재 이더말 증폭

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

의 탐지를위한 동결 건조 루프 - 매개 등온 증폭 시약의 개발

요약

더 많은 비디오 탐색

루프 매개 이소르말 증폭을 사용하여 관개물에서 피토프토라 캡시 검출

세대와의 멀티 표현형 높은 콘텐츠 상영

트리파노소마 크루지 또는 기타 병원성 유기체의 DNA 검출을 위한 즉시 사용 가능한 qPCR

적용 형광 공명 에너지 전달 (FRET)을하여 이펙터 전좌 효율을 검사합니다

모 세관 배열에 여러 개의 핵 산의 비주얼 검색

자원 제한 환경에서 실시간 이소레말 핵산 증폭 반응을 모니터링하는 오픈 소스 소형 플루오리미터

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