저자 족장, headmen와 모기 필드 샘플 컬렉션 중 협력의 unwaveing​​에 대한 마챠의 지역 사회에 빚을 졌네. 박사 덕 노리스과 리브가 켄트에서 소금의 프로토콜에 귀중한 전문 지식을 제공했습니다. 이 작품은 존스 홉킨스의 말라리아 연구소 파일럿 보조금 시스템에 의해 재정 지원되었다. 모기와 기생충 DNA 실험실 기준은 친절 MR4, 미국 유형 및 문화 컬렉션에 의해 기부되었습니다.

1. 모기 표본의 준비 해부 <ol><li> 현미경을 해부에 parafilm과 마운트의 작은 절단에 장소 모기. </li><li> 조직을 부드럽게하기 위해 탈 이온수의 드롭에서 다룹니다. </li><li> 따라서 복부 섹션에서 머리와 가슴을 nicking, 가슴과 복부 사이의 공동에 정확하게 모기 시체를 절개. </li><li> 모기 ID 번호 세부 사항 및 적절 본문 섹션 (; 머리와 가슴 부분의 &quot;HT&quot;중간 창자 섹션의 예 : &quot;m&quot;) 나타 내기 위해 표시와 함께 prelabeled 깨끗한 autoclaved 1.5 ML의 microcentrifuge 튜브에 각 해부하는 모기 섹션을 전송합니다. </li><li> parafilm을 취소하고 조심스럽게 옆에 모기을 처리하기 전에 70% 알코올 moistened Kimwipe과 현미경 무대를 닦아냅니다. </li></ol> 2. 모기 표본에서 DNA 추출 <ol><li> 샘플 튜브 및 해제에 가라 앉아 모기 섹션을 갈기 위해 사용 피펫 팁에 탈 이온수의 피펫 20 μliform 정지. </li><li> autoclaved 1X PBS / 부드러운 순간 vortexing하여 샘플 homogenate 및 혼합 1 %의 사포닌 솔루션 100 μl를 추가합니다. </li><li> 20 분 동안 실온에서 알을 품다. </li><li> 2 분에 20,000 XG에 원심 분리기와 표면에 뜨는 폐기하십시오. </li><li> 100 μl 1X PBS에 Resuspend 펠릿. </li><li> 2 분에 20,000 XG에서 다시 원심 분리기와 표면에 뜨는 폐기하십시오. </li><li> 부드러운 vortexing (5 초), 75 μl 멸균 탈 이온수에 resuspend 펠렛 및 탈 이온수 20 % w / v를 Chelex-100 수지 정지의 25 μl으로. </li><li> 분젠 버너에 골조 멸균 23G 피하 주사기 바늘을 사용하여 샘​​플 튜브의 뚜껑에 피어스 구멍. </li><li> 10 분에 떠있는 랙에 물 목욕에 넣어 익히지 샘플 중지 (또는 가열 블록에서). </li><li> PCR의 응용 프로그램에서 템플릿으로 사용할 수 prelabeled 스토리지 유리 병에 1 분 및 전송 다음의 DNA 솔루션, 20,000 XG에 원심 분리기. </li></ol> 3. Plasmodium falciparum Genotyping과 아노펠레스종. 분자 식별 <ol><li> 아노펠레스 종 10 달러를 파악하고 유전자형 P.에, DNA 품질 11을 확인하는 25 μl PCR 반응에 Chelex DNA 추출물 2.5 μl를 추가합니다 falciparum 12 중반 내장과 침샘에 염증. </li><li> 특히 P.에 대한 amplicon 수율을 극대화하려면 falciparum 감지는 PCR 분석에 (BSA) 1.5X 소 혈청 알부민이 포함되어 있습니다. 다음 반응 구성을 권장합니다 : (2.5 μl 템플릿, 0.25 μM 프리 머, 1.5 μM의 염화 마그네슘, 200 μM dNTPs, 1X PCR 버퍼, 1.5X BSA와 1.0U DNA 형성 촉매의 DNA 중합 효소를, 25 μl 권). </li><li> 추출물의 DNA 품질을 확인하려면 arthropod mitochondrial 니코틴 아마이드 아데닌 다이 뉴클레오타이드 탈수소 효소 (NADH) subunit 4 (ND4) 유전자 (프리 머 ND4FW [5&#39;-GTD야만 TTA TGA TTR CCT​​ AA-3 &#39;]와 ND4RV [5&#39;에게의 영역을 증폭 - CTT CGD CTT CCW ADW CGT TC-3 &#39;]; 제품 400bp) 11,13을 기대합니다. </li><li> 아노펠레스 감비아을 차별화하려면전자 SL. 형제 종 (An. gambiae SS와. arabiensis 만), intergenic 스페이서 (IGS) 지역, (에 SNPs 측면을 노릴 지역을 증폭 프리 머 UN [5&#39;-GTGTGCCCCTTCCTCGATGT-3 &#39;], GA [5&#39;-CTGGTTTGGTCGGCACGTTT - 3 &#39;], AR [5&#39;-AAGTGTCCTTCTCCATCCTA-3&#39;], 제품 390bp에게 gambiae SS, 315bp arabiensis) 10,11을 기대합니다. </li><li> 입력 P.에 대한 falciparum antifolate의 약물 저항 polymorphisms은 아미노산 codons 108, 51, 59, 기생충 dihydrofolate 환원 효소 (DHFR) 유전자 16 164 측면을 노릴 지역을 확대하려면 중첩 PCR을 수행 : 기본 원형 프리 머 : M1 [5&#39;-TTTATGATGGAACAAGTCTGC-3 &#39;]와 M5 [5&#39;-AGTATATACATCGCTAACAGA-3&#39;] 차 라운드 프리 머 : M3 [5&#39;-TTTATGATGGAACAAGTCTGCGACGTT-3 &#39;] F /과 [5&#39;-AAATTCTTGATAAACAACGGAAACCTTTTA-3&#39;] 또는 F [5&#39;-GAAATGTAATTCCCTAGATATGGAATATT-3] M4는 [5&#39;-TTAATTTCCCAAGTAAAACTATTAGAGCTTC-3 &#39;]). 또한 아미노산 codons 436, 437, 540, 581를 포함하는 지역을 증폭P.의 차 613 falciparum dihydropteroate의 synthetase (DHPS) 유전자 : 기본 원형 프리 머 R2 [5&#39;-AACCTAAACGTGCTGTTCAA-3 &#39;] R /과 [5&#39;-AATTGTGTGATTTGTCCACAA3&#39;]; 차 라운드 프리 머 J : [5&#39;- &#39;TGCTAGTGTTATAGATATAGGTGGAGAAAGC-3&#39;] K / [5&#39;-CTATAACGAGGTATTGCATTTATTGCAAGAA-3 &#39;]와 또는 K : K /과 [5&#39;-TGCTAGTGTTATAGATATAGGATGAGCATC-3 &#39;], 또는 L [5&#39;-ATAGGATACTATTTGATATTGATATTGGACCAGGATTCG-3 &#39;] L과 / [5&#39;-5TATTACAACATTTTGATCATTCGCGCAACCGG-3&#39;] 12. </li><li> 제조업체의 지침에 따라 30 μl 반응의 allele 특정 제한 효소 digestions,에 따라 4 μl amplicon은 antifolate 약물 저항 - 관련 polymorphisms 12을 감지합니다. </li><li> 제목 5 전기에 ethidium 브로마이드 - 스테인드 2퍼센트 아가로 오스 겔 (100 - 120V)의 차선 당 PCR amplicon (또는 제한 다이제스트의 15 μl)의 μl하고 UV transillumination에서 대역을 표시합니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

여기에 제시된 단순화 Chelex 방법은 품질 아노펠레스 균과 P.의 추출을 가능하게 다양한 PCR 응용 프로그램에 복종 할 의무가있는 모기 표본에서 falciparum의 DNA. 이 기술은 약제 내성 P.의 분자 말라리아 벡터 모기의 식별 및 감시를 위해 고용 수 있습니다 국가 말라리아 통제 프로그램에 대한 모기의 falciparum 대립 유전자. 단순화 Chelex 기술의 장점은 단순성, 적은 시약 및 따라서 비용, 안전 및 방법 (10) 아웃 소금 (2 시간 47 분, 야간 단계, 표 1)과 같은 표준 프로토콜보다 짧은 처리 시간을 (37 분) 등이 있습니다. 앞서 언급 한 장점과 최소한의 시약 요구 사항 (3 시약)은 현재의 표준 프로토콜 (10 시약, 표 1) 11,15에 비해, 일정한 시약 발병 국가 연구소로 단순화 Chelex 프로토콜은 특히 쉽게 만들공급 체인은 종종 유지하기가 어렵습니다. 방법의 한계는 표준 프로토콜처럼, 또한 모기 외피 16가 발생하는 것으로 알려져 PCR 억제제에 따라이라고합니다. 그러나, 이것은 쉽게 assays에 BSA를 포함하여 안심입니다. BSA는 성공적으로 다른 PCR 응용 프로그램 17,18의 억제제에 대한 증폭 증강으로 채용되었습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> Chelex-100, 50-100 건조 메쉬 </td><td> BioRad </td><td> # 143-2832 </td><td></td></tr><tr><td> 사포닌 </td><td> 시그마 </td><td> 142-7425 </td><td></td></tr><tr><td> BSA </td><td> 뉴 잉글랜드 Biolabs </td><td> B9001S </td><td></td></tr></tbody></table>

a simple chelex protocol for dna extraction from anopheles spp.

발병 국가들은 점점 자신의 제어 프로그램 1,2,3,4,5의 중요한 부분으로, 효율적인 타이핑, 신분증과 말라리아 기생충과 벡터 모기에 대한 감시를위한 분자 도구를 채택하고 있습니다. 작업에서 이러한 정확한 접근 방식의 지속 가능한 설립 말라리아 제어 및 지극히 검소한 시약 및 장비 요건을 제거 노력 간단하고 저렴한 방법을 강화하기 위해 연구 6,7,8 필수적입니다. 여기 현장 수집 모기 표본에서 말라리아 기생충 및 벡터 DNA를 추출하기위한 간단한 Chelex 기반 기술을 제시한다. 우리는 morphologically 72 아노펠레스 gambiae SL을 확인. pyrethrum 스프레이는 마챠에서 말라리아 연구소의 2,000 킬로미터 주변에서 가정의 객실이 수면에 기하여 캡처 156 모기에서. 절개는 72 아노펠레스 할머니를 위해 복부에서 머리와 가슴을 분리하는 후biae SL. 모기는 두 섹션은 개별적으로 1.5 ML의 microcentrifuge 튜브에 배치하고 탈 이온수 20 μl에 잠겨 있었다. 멸균 피펫 팁 사용하여 각 모기 섹션은 별도로 탈 이온수의 균일 한 현탁액으로 균질되었습니다. 다른 10 μl는 별도의 autoclaved 1.5 ML 튜브로 전송 동안 각 모기 섹션에서 다음의 homogenate 중, 10 μl이 유지되었다. 별도의 aliquots는 추출 프로토콜 9,10를 염장 단순화 Chelex하거나 표준에 의해 DNA 추출을 받게되었습니다. 이 DNA 추출 구 동안 단백질 침전 단계에 대한 유해 유기 용제 (예 : 페놀과 클로로포름 등) 대신에 높은 소금 농도를 사용하고 있기 때문에 아웃 소금의 프로토콜은 소위 광범위하게 사용됩니다. 추출물은 arthropod mitochondrial 니코틴 아마이드 아데닌 다이 뉴클레오타이드의 dehydroge을 대상으로 프리 머를 사용하여 PCR 증폭을위한 템플릿으로 사용되었습니다nase (NADH) subunit 4 유전자 (ND4)는 Plasmodium falciparum 감염 (12)의 입력을위한 DNA의 품질 11, 아노펠레스 gambiae의 형제 자매 종 (10)의 식별을위한 PCR과 중첩 된 PCR을 확인합니다. DNA 품질 (ND4)를 사용하여 비교 PCR은 기존의 아웃 소금의 프로토콜에 비해 Chelex 접근 방식 93 %의 민감도와 82 % 특이성을 보여 주었다. 감도 및 특이성의 상응하는 값은 P.에 각각, 형제 자매 종 식별 PCR, 92 %와 80 %를 사용하여 각각 100 %와 78%했다 falciparum 검출 PCR. Chelex 또는 일반은 세 PCR 응용 프로그램에서 프로토콜을 소금과 amplicon 신호를주는 샘플 비율에 큰 차이가 없었다. Chelex 접근 방식은 세 가지 간단한 시약 및 완료 37 분을 요구하면서 밤새 단계를 포함하여 10 가지 시약 및 2 시간, 47 분 &#39;처리 시간을 entailed 프로토콜을 소금. 우리의 결과는 일을 보여Chelex 방법에서 기존 아웃 소금의 추출과 비교하고 일정한 시약 공급 체인은 종종 유지하기 어려운 자원 제한 설정에서 간단하고 지속 가능한 방식으로 대체 할 수 있습니다.

모기 DNA 추출물 품질에 대한 PCR assays (그림 1), 아노펠레스의 arabiensis 분자 식별 (그림 2)와 P. 결과의 예 단순화 Chelex 방법이 훨씬 적은 단계 (표 1)에도 불구하고, 프로토콜 10을 소금 표준에 유사한 결과가 산출되는 것으로 falciparum 탐지 (그림 3)를 보여줍니다. 각각의 추출물에 비해 DNA의 품질은 놀라운 일이 아니다 그와 관련하여 샘플 긍정적 인 요금. gambiae의 형제 종뿐만 아니라 기생충 감염 요금은 McNemar의 치 평방 테스트 (그림 3)에 따라 통계적으로 차이가 없었다. 감도 (%)는로 계산 TP / (TP + FN) TP 사실 탐지를 나타냅니다과 FN 거짓 제외를 나타냅니다 * 100. 특이성 (%)가 TN /로 결정했습니다 (TN + FP) TN 사실 제외 나타냅니다과 FP 거짓 탐지를 나타냅니다 * 100. DNA 품질 (ND4) PCR은 설립으로 프로토콜을 소금의 황금 표준에 비해 Chelex 접근 방식 93 %의 민감도와 82 % 특이성을 보여 주었다. 감도 및 특이성의 상응하는 값은 P.에 각각, 형제 자매 종 식별 PCR, 92 %와 80 %를 사용하여 각각 100 %와 78%했다 falciparum 검출 PCR. 반응 혼합물에 BSA의 추가 프로토콜을 소금 간소화 Chelex과 정기적 모두, 때문에 PCR 저해제 (14)의 구조에 대한 PCR의 반응에 대한 일반적인 증가 (그림 3)에되었습니다. 그러나,이 증가는 Chelex 추출물 (P = 0.039)에 DNA 품질 (ND4 PCR)을 제외하고는 통계적으로 의미가 아니 었습니다. P.의 Allele 특정 제한 효소 소화 falciparum DHFR (또는 다른 대상 유전자) amplicon 약물 저항 대립 유전자 (; 침샘에 데이터가 표시되지 그림 4) 중간 창자와 침샘에 말라리아 감염의 genotyping 수 있습니다. <table border = &quot;1&quot;&gt; <tbody><tr><td colspan="2"> 간단한 Chelex 절차 </td><td colspan="2"> 표준 절차를 염장 </td></tr><tr><td> 단계 </td><td> 시약 </td><td> 단계 </td><td> 시약 </td></tr><tr><td><ol><li> 1.5 ML의 microfuge 관에 시료를 추가하고 1X PBS / 1 %의 사포닌 솔루션 (2 분) 100 μl에 균질화. </li><li> 20 분 동안 실온에서 둡니다. </li><li> 2 분에 20,000 XG에서 회전하고 표면에 뜨는 폐기하십시오. </li><li> 1X PBS 100 μl를 추가하고 잠시 (3 초) vortexing으로 부드럽게 섞는다. </li><li> 2 분에 20,000 XG에서 회전하고 표면에 뜨는 폐기하십시오. </li><li> 탈 이온수 75 μl 멸균 탈 이온수 20 % w / v를 Chelex의 25 μl를 추가합니다. </li><li> 10 분 동안 뜨거운 무균 23G 피하 주사기 바늘과 종기 튜브 내용을 사용하여 샘​​플 튜브의 뚜껑에 피어스 구멍. </li><li> 2 microfuge 튜브를 스핀1 분에 0,000 XG. </li><li> 사용 때까지 -20 ° C (25 μl PCR 반응에 2.5 μl)에서 무균 prelabeled 유리 병 및 매장 DNA 솔루션으로 표면에 뜨는을 전송합니다. </li></ol></td><td> PBS 사포닌 Chelex-100 구슬 </td><td><ol><li> 1.5 ML의 microfuge 관에 시료를 추가하고 1 % DEPC (2 분) 100 μl 벤더 버퍼 *에 균질화. </li><li> 1 시간에 65 ° C에서 알을 품다. </li><li> 15 μl 추운 8 M의 칼륨 아세테이트을 추가합니다. 부드럽게 섞은 후 45 분에 얼음에 품다. </li><li> 새로운 1.5 ML 튜브에 표면에 뜨는 10 분 및 전송을위한 20,000 XG에서 microfuge 튜브에 샘플을 봐. </li><li> 100 % 에탄올 250 μl를 추가하고 반전 관으로 잘 섞는다. </li><li> 5 분을 위해 RT에서 샘플을 품다. </li><li> 15 분에 20,000 XG에서 샘플을 봐. </li><li> 기음과 (30m 튜브에 펠렛가 완전히 건조두고, 표면에 뜨는 폐기)에 있습니다. </li><li> 4에 밤새 0.1X SSC + RNAse (10 μg / ML)의 20 μl에 Resuspend 펠릿 ° C. </li><li> 사용 때까지 -20 ° C 80 μl DEPC 물과 매장을 추가합니다. </li></ol></td><td> Diethylpyrocarbonate (DEPC) * 벤더 버퍼 : 0.1 M NaCl 0.2 M의 자당 0.1 M 트리스 - HCL 0.05 M EDTA (에틸렌 다이아 민 테트라 초산), 산도 9.1 DEPC 물에 0.5 % SDS 8 M의 칼륨 아세테이트 에탄올 0.1X 식염 나트륨 구연산 (SSC) 버퍼 RNAse (10 μg / ML) </td></tr><tr><td colspan="2"> 총 소요 시간 : 37 분 </td><td colspan="2"> 총 소요 시간 : 2 시간 47 분, 플러스 하루 아침에 </td></tr></tbody></table> 표 1. 간단한 Chelex 프로토콜에 대한 시약 및 시간 요구 사항 단계 비교에 의해 단계 서ARD는 모기 표본에서 DNA 추출을위한 방법을 소금. <img alt="그림 1" src="/files/ftp_upload/3281/3281fig1.jpg" /> 그림 1. ND4 mitochondrial PCR의 단순화 Chelex &quot;C&quot;에서 DNA 품질의 비교 및 아노펠레스의 arabiensis 필드 샘플에 &quot;M&quot;추출물을 소금의 표준입니다. NC, 음성 제어, M1 및 C1, M2와 C2, M3 및 C3, 그리고 M4와 C4는 아웃 소금과 같은에서 Chelex 추출 짝입니다. arabiensis의 모기 표본. L, 100 BP의 DNA 사다리. <img alt="그림 2" src="/files/ftp_upload/3281/3281fig2.jpg" /> 그림 2 아노펠레스의 arabiensis에 대한 분자 식별 PCR C1과 M1, C2 및 M2, C3와 M3, C4와 M4는 &quot;M&quot;을 소금과 같은 모기 표본에 대한 Chelex &quot;C&quot;DNA 추출물을 단순화에서 각각의 amplicon을 표시,.. AR, 아노펠레스 arabiensis긍정적 인 제어, L, 100 BP의 DNA 사다리. <img alt="그림 3" src="/files/ftp_upload/3281/3281fig3.jpg" /> 그림 3. Chelex과 아노펠레스 arabiensis (AR) 10, arthropod NADH 탈수소 효소 유전자 4 (ND4) DNA 품질 테스트 11, 및 antifolate 약물 저항 P의 분자 식별 PCR assays와 함께 모기 필드 샘플에 대한 DNA 추출물을 소금에 탐지 검색 . falciparum DHFR genotyping 12 (F-M4). 반응 믹스에 BSA와 함께 또는없이 실행 assays에 대한 결과가 표시됩니다. McNemar의 치 평방 테스트는 DNA로부터 긍정적 인 PCRs의 비율의 차이는 간단 Chelex에서 추출 및 프로토콜을 소금 표준 통계적으로 의미가 있었다 여부를 결정하는 데 사용되었다. <img alt="그림 4" src="/files/ftp_upload/3281/3281fig4.jpg" /> <stroNG&gt; 그림 4. genotyping P.의 단순화 Chelex 프로토콜의 응용 약물 저항 대립 유전자에 대한 모기의 falciparum 감염 (중간 창자 데이터 참조). P.의 코돈 108 측면을 노릴 amplicon에 BstN 나는 소화 falciparum DHFR 유전자 12 모기 샘플 (; 중순 내장 데이터가 표시 M1-M5)에 cycloguanil 방지 S108T의 돌연변이를 보여줍니다. U, 소화되지 않은 522 BP amplicon, FCR3, 실험실 표준 P. S108T를 들고 falciparum 긍정적 인 제어 클론, K1, P. falciparum 실험실 표준 cycloguanil에 민감한 S108N을 들고 클론 부정적인 제어, L, 100 BP의 DNA 사다리.

Watch this Scientific Journal Video about A Simple Chelex Protocol for DNA Extraction from Anopheles spp. at JoVE.com

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

모기 표본의 품질 말라리아 기생충 및 벡터 DNA를 추출 할 수 신속하고 저렴한 방법은 설명되어 있습니다. 간단한 방법은 모기 중간 창자와 침샘 단계에서 말라리아 기생충의 genotyping,뿐만 아니라의 분자 식별, Chelex 수지의 킬레이트 특성에 수 대문자로 표기<em&gt; 아노펠레스</em&gt; PCR에 의해 종을 형제.

에서 DNA 추출을위한 간단한 Chelex 프로토콜<em&gt; 아노펠레스</em&gt; 종.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

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A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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54.6K 조회수.  Malaria Institute at Macha. 모기 표본의 품질 말라리아 기생충 및 벡터 DNA를 추출 할 수 신속하고 저렴한 방법은 설명되어 있습니다. 간단한 방법은 모기 중간 창자와 침샘 단계에서 말라리아 기생충의 genotyping,뿐만 아니라의 분자 식별, Chelex 수지의 킬레이트 특성에 수 대문자로 표기 아노펠레스 PCR에 의해 종을 형제.

비디오: A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

옐로 피버 모스키토에서 Hemocytes를 수집 및 스테 이닝을위한 프로토콜 Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IgY 기술 : 폴리에틸렌 글리콜의 노른자위에서 치킨 항체의 추출 (PEG) 강수량

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

루트 - 매듭 Nematodes과 높고 낮은 처리량 화면 Meloidogyne SPP.</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

의 혈소판 응집 중재를위한 간단한 프로토콜<em&gt; 변형체 falciparum의</em자원별로 설정에서&gt; - 감염된 적혈구

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

침투 림프구의 분석을위한 비 효소 해리 신선한 인간의 조직에 대한 간단하고 빠른 프로토콜

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

간단하고 효율적인 접근 방법은 돌연변이 백시 니아 바이러스 벡터를 구축하기

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

새로운 바이러스 게놈을 발견 감염 된 잎에서 RNA 분석 원유 Virion 추출에 DNA의 결합 분석

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

전체 혈액 샘플에서 바이러스의 검출, 안전, 신속 하 고 간단한 수동 침대 핵 산 추출

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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 ...

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

This protocol provides a simple, cost-effective DNA isolation method for the analysis of murine gut microbiota alterations during the development of autoimmune disease.

간단하고 비용 효율적인 DNA 분리 방법을 사용한 루푸스 경향 마우스의 분변 미생물 역학 분석

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that ...

Methods Article

에서 DNA 추출을위한 간단한 Chelex 프로토콜