저자는 월위한 표준 및 제어 반응의 종류 선물 질병 통제 예방을위한 국립 Calicivirus 연구실 센터 (CDC)에 감사 드리고, 그리고 시약을 제공 존스 홉킨스에​​서 공중 보건 학부의 실험실 것이다.

1. 대변​​ 샘플 <ol><li> 대변​​ 샘플은 RNA를 유지하기 위해 냉동 보관해야합니다. 10 % 배설물 현탁액을 하시려면 해동 대변 샘플의 약 0.1 g을 가지고 PBS 1 개 ML을 완료합니다. </li><li> 피할 200 μl의 나누어지는은 동결 융해 반복했다. -70 ° C.에 aliquots를 저장 </li><li> 영유권 및 추출에 사용하기 전에 10 분 동안 4천그램에서 aliquots을 원심 분리기. </li></ol> 2. 구아니딘 및 실리카로 추출을위한 실리카 입자의 작성 <ol><li> RT에서는 이산화 규소 30 G를 중지하고 최대 250 ML에 DH 2 O와 완료합니다. </li><li> 침전의 24 시간 후에 흡입에 의해 뜨는의 215 ML을 제거합니다. 최대 250 ML에 DH 2 O를 추가하고 흔들림으로 실리카 펠렛을 일시 중지합니다. </li><li> 또 24 시간 후에 흡입에 의해 뜨는을 제거하고 pH를 2.0으로 실리카 현탁액의 산도를 조정, 염산 (HCL)을 사용합니다. 유리 봇에서 나누어지는 실리카 현탁액을tles 및 압력솥. </li></ol> 3. 구아니딘 및 실리카로 추출을위한 L6 버퍼의 작성 <ol><li> RT에서, 0.1 M 트리스 염산염, 산도 6.4, 그리고 0.2 M EDTA (에틸렌 다이아 민 테트라 초산), 산도 8.0 용액 중 11 ML의 50 ML에 X-100 guanidinium thiocyanate의 60 G (GuSCN)와 트라 이튼 1.3 g을 용해하여 L6 버퍼를 준비합니다. </li></ol> 4. 구아니딘 및 실리카로 추출을위한 L2 버퍼의 작성 <ol><li> RT에서, 0.1 M 트리스 염산염, 산도 6.4의 150 ML에 guanidinium thiocyanate 180 G (GuSCN)을 해소하여 L2 버퍼를 준비합니다. </li></ol> 5. 추출 절차 <ol><li> 각 추출에 월 긍정적인 대변 샘플은 긍정적인 제어 및 DEPC 처리 물로, 부정적인 컨트롤로 포함되어야합니다. </li><li> 마이크로 원심 튜브에 다음을 섞어, 버퍼 L6, 실리카 입자 20 μL 1 ML, 그리고 10 % 배설물 추출물 서스펜션 200 μL를 추가합니다. 와동의 간략 15 마일을 위해 RT에두고N. </li><li> 10 s에 대한 6천그램에서 현탁액을 원심 분리기와 70 %의 에탄올을 가진 두 세차장 및 아세톤으로 한 번에이어서, 두 버퍼의 L2의 1ml와 펠렛을 씻는다. 5 분 동안 56에서 건조 가열 블록 ° C로 펠렛을 건조합니다. </li><li> RNase가없는 DH 2 0 50 μL를 추가하여 실리카 펠렛의 하이드 RNA. 15 분 동안 RNasin 1 μL, 56에서 반전 튜브 및 부화에 의한 믹스 ° C를 추가합니다. </li><li> 탁상 원심 분리기에서 전속력으로 3 분 동안 원심 분리기와 RNA를 포함하는 표면에 뜨는 40 μL를 수집합니다. </li><li> Nanodrop 2000 분광 광도계 (써모 과학)을 사용하여 압축을 푼 RNA 샘플을 계량. 그렇다면 -70에서 보관 ° C를 이용 때까지 최대 6 개월까지. (참고 : 손상 총 RNA를 유지하기 위해 더 좋은 방법은 cDNA로 변환합니다.) </li></ol> 6. 상업 RNA QIAamp 바이러스성 RNA Kit를 사용하여 대체 추출 전체 지침은 책자 prov에서 발견되는QIAGEN 키트 ided. 다음과 같이 간단히 절차는 다음과 같습니다 <ol><li> 버퍼 AVL의 Pipet 560의 μL은 1.5 ML 튜브로 캐리어 RNA를 포함하고, 10 %의 대변 현탁액 140 μL를 추가합니다. 15 초위한 펄스 vortexing하여 섞는다. 10 분 동안 RT에서 품어. </li><li> 15 초 동안 펄스 vortexing에 의해 시료와 혼합으로 에탄올의 560 μL (96~100%)를 추가 후 간단히 원심 분리기. </li><li> 2 ML 회수 관에 배치 스핀 컬럼이 솔루션의 630 μL을 적용합니다. 6천g에서 1 분 동안 원심 분리기, 그리고 깨끗한 두 ML 관에 스핀 컬럼을 넣으십시오. </li><li> 1 분 용 6천그램에서 버퍼 AW1 및 원심 분리기 500 μL로 묶인 RNA를 포함하는 컬럼을 씻으십시오. 깨끗한 두 ML 수집 관에서 개최 칼럼. </li><li> 3 분은 최고 속도 (20,000 G 또는 14,000 RPM)에서 500 버퍼 AW2의 μL와 원심 분리기로 열을 씻으십시오. </li><li> 깨끗한 1.5 ML 마이크로 원심 분리기 튜브의 장소 스핀 컬럼은 40 μL DEPC-처리된 물을 추가하고 전속력으로 RNA를 elute. 에 반복eluted RNA의 최종 80 μL를위한 CE. </li><li> Nanodrop 2000 분광 광도계 (써모 과학)을 사용하여 압축을 푼 RNA 샘플을 계량. 그렇다면 -70에서 보관 ° C를 이용 때까지 최대 6 개월까지. (참고 : 손상 총 RNA를 유지하기 위해 더 좋은 방법은 cDNA로 변환합니다.) </li></ol> 7. 한 단계 실시간 특정 Taqman 프로브와 RT-PCR에 의해 추출된 RNA의 노로 바이러스의 검출 악기 StepOnePlus 리얼 타임 PCR 시스템 (응용 Biosystems). 방법론 <ol><li> 닦아내기 및 기타 잠재적인 RNase 오염을 제거 행방불명 RNase와 함께 모든 작업 표면, pipettes, 그리고 원심 분리기를 청소합니다. </li><li> Pipet 월 GI과 GII 심사 마스터는 표 1에 따라 혼합. Primers 및 Taqman 프로브는 표 2에 나열되어 있습니다. </li><li> 각 반응 튜브에 맞는 마스터 믹스 나누어지는 10 μL. </li><li> undiluted 미지 시료의 5 μL를 추가하십시오해당 반응 우물에 각각 RNA, DEPC-처리된 대조군과 같은 물, 혹은 GI GII 긍정적인 제어 RNA. 모든 샘플은 중복으로 실행해야합니다. 각각 300 μg / μL와 500 μg / μL의 농도로 긍정적인 컨트롤과 기준은 질병 통제 예방을위한 국립 Calicivirus 연구실 센터 (CDC)에 의해 제공되었다. </li><li> 10 초 동안 6,000g에 반응 판을 원심 분리기. </li><li> 실험 등록 정보 화면에서, 실행을위한 실험의 종류를 정의하고 선택합니다. TaqMan의 시약은 시약의 종류 및 그 사전 PCR 증폭 읽기와 선택으로 표시하고 있는지 확인하십시오. </li><li> 표 3에서 열 프로파일을 사용하여 15 μL 반응을 실행합니다. </li><li> 결과를 분석. </li></ol> 8. 기존의 RT-PCR과 장면을 사용하여 월 GI과 GII의 Genotyping (이것은 일반적이고 널리 사용되는 방법 이니 그것은이 동영상에서 언급하지 않은 것.) 악기는. Biosystems StepOne 및 Quantitec 템플릿과 StepOnePlus 실시간 PCR 시스템을 적용했습니다. 방법론 <ol><li> genotyping 전에 샘플 genogroup (GI 또는 GII)은 RT-PCR은 위에서 설명한 심사에 따라 결정됩니다. GI 월 RNA는 GII genotyping 마스터 믹스와 GI의 genotyping 마스터 믹스와 GII 월 RNA와 증폭되어야한다. </li><li> Pipet 월 GI과 GII genotyping 마스터는 표 4에 따라 혼합. Primers GI SKF / SKR 및 G2 SKF / SKR은 표 5에 나열되어 있습니다. </li><li> 0.2 ML 반응 튜브에 적합한 마스터 믹스 나누어지는 45 μL. </li><li> 각 부정적인 제어 튜브에 DEPC-처리된 물에서 5 μL, 그리고 사전 스크린, 월 (GI 및​​ / 또는 GII) 해당 반응 튜브에 긍정적인 RNA의 5 μL를 추가합니다. </li><li> 표 6에 온도 프로파일을 사용하여 50 μL 반응을 실행합니다. </li><li> PCR 제품 contai 보내기적어도 100은 증폭 capsid 지역의 μL / NG 정화 및 시퀀싱을위한 회사 Macrogen하기 닝. (시퀀싱 당 9,700 위대한 세네카 Hwy. 락빌, 메릴랜드 20850 및 샘플 당 $ 10). </li></ol> 9. 대표 결과 <img src="/files/ftp_upload/3232/3232fig1.jpg" alt="그림 1" /> 그림 1은 Taqman diarrheal 아이들의 대변 샘플에서 추출한 RNA 분석에 사용되는 원 - 스텝 RT-PCR에서 대표적인 결과를 보여줍니다. 긍정적인 샘플에 대한 문턱주기 (CT)은 GII를위한 GI와 CT 39 이하 CT 37 같음에서 설정되었습니다. 긍정적인 컨트롤을위한 CT-값은 각각 27, GI과 GII위한 ORF1-ORF2 교차점 18 미만이어야하는 것으로 확인되었다. <a href="http://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank">큰 그림을 보려면 여기를 누르십시오</a> . <img src="/files/ftp_upload/3232/3232fig2.jpg" alt="그림 2" /> 그림 2 <table border="1"><tbody><tr><td> 마스터 믹스 </td><td> 최종 Conc </td><td> 볼륨 (μL) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 2.875 </td></tr><tr><td> Quantitect RT-PCR 마스터 믹스 </td><td> 1X </td><td> 6.250 </td></tr><tr><td> RT 믹스 </td><td> 1X </td><td> 0.125 </td></tr><tr><td> 50 μm의 예지 자의 R 프라이머 </td><td> 1 μm의 </td><td> 0.250 </td></tr><tr><td> 50 μm의 예지 F 조의 입문서 </td><td> 일μm의 </td><td> 0.250 </td></tr><tr><td> 10 μm의 링 프로브 </td><td> 1 μm의 </td><td> 0.125/0.250 * </td></tr><tr><td></td><td></td><td> 10.00 </td></tr></tbody></table> 각 프로브의 0.125 μL가 GII 시험에 사용되는 동안 각 프로브의 * 0.250 μL은 GI 테스트에 사용되었다. 심사 GI과 GII위한 표 1. Qiagen Quantitect 마스터 믹스 (트루히요 외., 2006) 7. <table border="1"><tbody><tr><td> 이름 </td><td> 제노 - 그룹 </td><td> 사용 </td><td> 시퀀스 (5 &#39;3&#39;) </td></tr><tr><td> 코그 1R </td><td> 노병 </td><td> 뇌관 </td><td> CTT AGA CGC CAT CAT CAT TYA C </td></tr><tr><td> 코그 1 층 </td><td> 노병 </td><td> 뇌관 </td><td> CGY TGG ATG CGN TTY CAT 조지아 </td></tr><tr><td> 코그 2R </td><td> GII </td><td> 뇌관 </td><td> TCG ACG CCA TCT TCA TTC ACA </td></tr><tr><td> 코그 2 층 </td><td> GII </td><td> 뇌관 </td><td> 자동차 GAR BCN ATG TTY AGR TGG ATG AG </td></tr><tr><td> 반지 1A </td><td> 노병 </td><td> 탐침 </td><td> 우리 식구들-AGA TYG CGA TCY CCT GTC CA-BHQ-1 </td></tr><tr><td> 링 1B </td><td> 노병 </td><td> 탐침 </td><td> 우리 식구들-AGA TCG CGG TCT CCT GTC CA-BHQ-1 </td></tr><tr><td> Ring2-TP </td><td> GII </td><td> 탐침 </td><td> 우리 식구들-TGG 개그 GGC GAT CGC AAT CT-BHQ-1 </td></tr></tbody></table> 표 2. 프라이머와 genogroups I, II (Kageyama 외.위한 실시간 정량 RT-PCR에 사용되는 프로브 oligonucleotides,2003) 8. <table border="1"><tbody><tr><td> 단계 </td><td> 온도 (° C) </td><td> 시간 (분) </td><td></td></tr><tr><td> 일 </td><td> 50 </td><td> 30:00 </td><td> cDNA 합성 </td></tr><tr><td> 2 </td><td> 95 </td><td> 15시 </td><td> HotStart DNA 형성 촉매 효소의 활성화 </td></tr><tr><td> 3 </td><td> 95 </td><td> 0시 15분 </td><td></td></tr><tr><td></td><td> 60 </td><td> 1시 </td><td> 45X주기 </td></tr></tbody></table> 테이블3.를위한 써멀 프로파일 한 단계 Taqman 실시간 RT-PCR (트루히요 외., 2006) 7. <table border="1"><tbody><tr><td> 마스터 믹스 </td><td> 최종 Conc </td><td> 볼륨 (μL) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 29.50 </td></tr><tr><td> 5 배 Qiagen RT-PCR 버퍼 </td><td> 1X </td><td> 10.00 </td></tr><tr><td> 10 MM dNTP 믹스 </td><td> 0.4 MM </td><td> 2.00 </td></tr><tr><td> 효소 믹스 </td><td></td><td> 2.00 </td></tr><tr><td> 40 U / μL RNAsin </td><td> 20 U / μL </td><td> 0.50 </td></tr><tr><td> 10 μm의 SKF </td><td> 0.1 μm의 </td><td> 0.50 </td></tr><tr><td> 10 μm의 제품 SKR </td><td> 0.1 μm의 </td><td> 0.50 </td></tr></tbody><tbody><tr><td></td><td></td><td> 45.00 </td></tr></tbody></table> 표 4. Qiagen genotyping GI과 GII를위한 원 - 스텝 RT-PCR 마스터 믹스. <table border="1"><tbody><tr><td> 이름 </td><td> 제노 - 그룹 </td><td> 사용 </td><td> 시퀀스 (5 &#39;3&#39;) </td></tr><tr><td> G1SKF </td><td> 노병 </td><td> 뇌관 </td><td> 5&#39;-CTG C​​CC GAA TTY GTA AAT GA - 3 </td></tr><tr><td> G1SKR </td><td> 노병 </td><td> 뇌관 </td><td> 5&#39;-CCA ACC 차 CCA TTR 전술 - &#39;3 </td></tr><tr><td> G2SKF </td><td> GII </td><td> 뇌관 </td><td> 5&#39;-CNT GGG AGG GCG ATC GCA - 3 </td></tr><tr><td> G2SKR</td><td> GII </td><td> 뇌관 </td><td> 5&#39;-CCR CCN GCA TRH CCR TTR TA CAT-3 </td></tr></tbody></table> 표 5. Primers는 Capsid 지역의 지역 C (코지마 외., 2002)의 증폭에 사용 5. <table border="1"><tbody><tr><td> 단계 </td><td> 온도 (° C) </td><td> 시간 (분) </td><td></td></tr><tr><td> 일 </td><td> 60 </td><td> 30:00 </td><td> cDNA 합성 </td></tr><tr><td> 2 </td><td> 96 </td><td> 15시 </td><td> HotStart DNA 형성 촉매 효소의 활성화 </td></tr><tr><td> 3 </td><td> 94 </td><td> 0시 3분0 </td><td></td></tr><tr><td></td><td> 52 </td><td> 1시 </td><td> 40X주기 </td></tr><tr><td></td><td> 72 </td><td> 0시 반 </td><td></td></tr><tr><td> 4 </td><td> 72 </td><td> 10시 </td><td> 가열 냉각 </td></tr></tbody></table> 표 6. Taqman 원 - 스텝 RT-PCR을위한 열 프로필입니다.

<ol>
	<li>Medici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+epidemiology+of+Norovirus+infections+in+sporadic+cases+of+viral+gastroenteritis+among+children+in+Northern+Italy.">Molecular epidemiology of Norovirus infections in sporadic cases of viral gastroenteritis among children in Northern Italy.</a> L. Medical Virology. 78, (2006).</li><li>Vidal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+recombinant+Norovirus+causing+outbreaks+of+gastroenteritis+in+Santiago,+Chile.">Novel recombinant Norovirus causing outbreaks of gastroenteritis in Santiago, Chile.</a> J. Clinica Microbiology. 4, (2006).</li><li>Xavier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+caliciviruses+associated+with+acute+infantile+gastroenteritis+in+Salvador,+an+urban+center+in+Northeast+Brazil.">Detection of caliciviruses associated with acute infantile gastroenteritis in Salvador, an urban center in Northeast Brazil.</a> Braz. J. Med. Biol. Res. 42, (2009).</li><li>Boom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+simple+method+for+purification+of+nucleic+acids.">Rapid and simple method for purification of nucleic acids.</a> J. Clin. Microbiol. 28, 495-503 (1990).</li><li>Kojima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genogroup-specific+PCR+primers+for+detection+of+Norwalk-like+viruses.">Genogroup-specific PCR primers for detection of Norwalk-like viruses.</a> J. Virol. Methods. 100, 107-114 (2002).</li><li>Mattison, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+comparison+of+two+norovirus+ORF2-based+genotyping+protocols.">Multicenter comparison of two norovirus ORF2-based genotyping protocols.</a> J. Clin. Microbiol. 47, 3927-3932 (2009).</li><li>Trujillo, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+TaqMan+real-time+reverse+transcription-PCR+for+rapid+detection,+quantification,+and+typing+of+norovirus.">Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus.</a> J. Clin. Microbiol. 44, 1405-1412 (2006).</li><li>Kageyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+broadly+reactive+and+highly+sensitive+assay+for+Norwalk-like+viruses+on+real-time+quantitative+RT-PCR.">A broadly reactive and highly sensitive assay for Norwalk-like viruses on real-time quantitative RT-PCR.</a> J. Clin. Microbiol. 41, 1548-1557 (2003).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

대변​​ 샘플로부터 핵산을 분리해위한 방법을 사내에 경제를 사용하여, 우리는 Qiagen의 상업 QIAmp 바이러스성 RNA 키트와 마찬가지로 동일한 결과를 얻을, 함께 TaqMan RT-PCR 저희 연구실에서 개발된들과 우리 월 광범를 감지할 수 있습니다 GI과 GII genogroup에 속하는 genotypes. 지역 C 프로토콜의 최근 출간은 78 % 6의 genotyping 속도를 보도했다. 현대 월의 종자의 다양성 이전 년 동안 증가되어 있?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 구아니딘의 isothiocyanate </td><td> 시그마 - Adrich </td><td> G9277 </td><td></td></tr><tr><td> 트리스 HCL </td><td> 시그마 - 올드 리치 </td><td> T5941 </td><td></td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 시그마 - 올드 리치 </td><td> E5134 </td><td></td></tr><tr><td> 규토 </td><td> 시그마 - 올드 리치 </td><td> S5631 </td><td></td></tr><tr><td> 트리톤 X 100 </td><td> BDH 화학 </td><td> 14,530 </td><td></td></tr><tr><td> Diethylpyrocarbonate </td><td> 시그마 - 올드 리치 </td><td> D-5758 </td><td></td></tr><tr><td> 바이러스성 RNA 미니 키트 (250) QIAamp </td><td> QIAGEN </td><td> 52,906 </td><td></td></tr><tr><td> QuantiTEC 프로브 RT-PCR 키트 (200) </td><td> QIAGEN </td><td> 204,443 </td><td></td></tr><tr><td> Qiagen 한 단계 RT-PCR 키트 (200) </td><td> QIAGEN </td><td> 210,212 </td><td></td></tr><tr><td> Rnase 억제제 2,000 단위 </td><td> A.Biosystems </td><td> N808-0119 </td><td> 2000 unids / 유리병 </td></tr><tr><td> 비 스틱 Rnae 프리 Microfuge 튜브 </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> UltraPure 아가로 오스 1,000 </td><td> Invitrogen </td><td> 16550-100 </td><td></td></tr></tbody></table>

detection and genogrouping of noroviruses from children's stools by taqman one-step rt-pcr

Noroviruses (NoVs)는 모든 연령층의 사람에있어서 세계적으로 산발적 급성 위장염의 발생의 주요 원인입니다. 그들은 Rotavirus의 그것과 유사한 공중 보건에 미치는 영향과 아동 입원의 중요한 원인입니다. NoVs 좋은 유전자 다양성의 RNA 바이러스이며 새로운 변종의 지속적인 모습이 있습니다. 다섯 genogroups 인식되며, GI과 GII들의 많은 genotypes과 인간의 감염에 대한 가장 중요한되고 subtypes와 함께. 그러나,이 두 genotypes의 진단은 진단과 치료를 지연, 문제가 남아 있습니다. 1, 2, 3 대변​​ 표본에서 RNA 추출에 가장 일반적으로 사용되는 방법은 Qiagen에서 QIAmp 바이러스성 RNA 상용 키트입니다. 이 메서드는 버퍼, 실리카 겔 막의 바인딩 속성을 결합하여 그 제어 RNases 및 microspin의 속도와 함께 컬럼으로 RNA의 최적의 바인딩을 제공합니다. 이 방법은 간단하고 빠르고 신뢰성이 있으며 카입니다제조 업체에서 제공한 설명에 자세히되는 몇 가지 단계에 뻔했어. 노로 바이러스는 설사의 가장 흔한 원인으로서 rotavirus에게만 초입니다. 노로 바이러스 진단은 설사의 pathogenesis에 대한 모든 연구에서뿐 아니라 발병이나 개별 설사 케이스에서 사용할 수 있어야합니다. 현재는 그러나 노로 바이러스 진단은 진단 간단한 방법의 부족으로 인해 단 몇 센터로 제한됩니다. 이 지연 진단 및 치료 1, 2, 3. 또한, 국가 사이와 사이의 비용과 부식성 버퍼의 규제로 인해 교통이 병참 문제를 포즈 이러한 제조 키트를 사용하십시오. 따라서이 프로토콜에 우리는 원래 붐을 기반으로 대안, 경제, 사내 방법을 설명 외 guanidinium thiocyanate의 안티 nuclease 특성과 함께 실리카 입자의 핵산 바인딩 속성을 사용합니다. 방법 4 . <p class=대변​​ 표본, 다른 목표를 활용하여 여러 RT-PCR 프로토콜에서 NoVs의 분리의 탐지 및 genogrouping (GI과 GII)은 &quot;jove_content&quot;&gt;이 개발되었습니다. 합의는 높은 감도, 특이성과 높은 처리량과 편리한 사용과 재현성을 결합하므로 TaqMan 화학을 사용하여 RT-PCR이 진단에 가장 적합한 분자 기법 될 것입니다. 여기 열린 독서 프레임 1 (ORF1) ORF2 접합 영역을 대상으로 분석을 설명, 진단 따라서 가장 보존 월의 게놈의 지역과 가장 적합합니다. 추가로 유전자 분석 capsid (지역 C) 원래 코지마 외 의해 설명되어 primers를 사용 5.의 주요 단백질에서 높은 변수 N-터미널 - 껍질을 대상으로 기존의 RT-PCR의 경우 상세하게됩니다. 종래의 PCR에서 PCR 제품의 시퀀싱하면 GI과 GII의 genogroups에 속하는 genotypes의 차별을 가능하게합니다.

Watch this Scientific Journal Video about Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR at JoVE.com

Detection and Genogrouping of Noroviruses from Children&#039;s Stools By Taqman One-step RT-PCR

primers와 열린 독서 프레임 1 (ORF1) ORF2 접합 영역입니다 노로 바이러스의 게놈 중 가장 보존 지역에 대한 구체적인 TaqMan 프로브를 이용하여 아이들의 stools에서 탐지 및 노로 바이러스의 분리의 genogroup 식별을위한 원 - 스텝 RT-PCR 분석 설명했다. 비상업적인 비용 효율적인 RNA 추출 방법을 자세히 설명하고 있습니다.

Taqman함으로써 어린이 Stools에서 Noroviruses의 감지 및 Genogrouping 한 단계 RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

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Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

17.1K 조회수.  Universidad Peruana Cayetano Heredia. primers와 열린 독서 프레임 1 (ORF1) ORF2 접합 영역입니다 노로 바이러스의 게놈 중 가장 보존 지역에 대한 구체적인 TaqMan 프로브를 이용하여 아이들의 stools에서 탐지 및 노로 바이러스의 분리의 genogroup 식별을위한 원 - 스텝 RT-PCR 분석 설명했다. 비상업적인 비용 효율적인 RNA 추출 방법을 자세히 설명하고 있습니다.

비디오: Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

정량 실시간 PCR (qPCR)에 의한 전립선 종양에 MicroRNA 감지

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

연구

의학

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

암 치료 모니터링을위한 Cerenkov 발광 이미징 (CLI)

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

마우스 모델에서 하대 정맥 끼어 이식의 주입

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

질병 모델링을위한 근육 조직 검사에서 환자 유래 세포주의 분리 및 멸화

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

전체 조직과 조직 검사에서 인간 상피 Enteroids과 Colonoids 설립

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

췌장 덕트 주입: 효과적이고 선택적 약물 및 바이러스 성 전달 방법

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

비 방사성 L-azidohomoalanine 라벨 방법에 의해 초기 단백질 합성 분석에 대 한 기본 마우스 Hepatocytes의 절연

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

아이 들에서 금속 이물질 섭취에 대 한 휴대용 금속 탐지기 검사

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

VASP 인 산화를 측정 하 여 혈액에 산화 질소의 혈소판 기반 탐지

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

쥐에서 히알로이드 혈관의 평가 및 특성화

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...