Yazarlar şefleri, muhtarları ve sivrisinek alanında örnek koleksiyonları sırasında işbirliği onların unwaveing ​​için Macha toplulukları borçluyuz. Dr Doug Norris ve Rebekah Kent üzerinden tuzlama protokol biçilmez bir uzmanlık sundu. Bu çalışma, Johns Hopkins Sıtma Araştırma Enstitüsü pilotu hibe sistemi tarafından finanse edildi. Sivrisinek ve parazit DNA laboratuvarında standartlara nazik MR4, Amerikan Tip Kültür &amp; Koleksiyon tarafından bağışlandı.

1. Sivrisinek Örneklerinin Hazırlık Diseksiyon <ol><li> Mikroskop diseksiyon üzerine parafilm ve montaj küçük bir kesim üzerinde yer sivrisinek. </li><li> Doku yumuşatmak için deiyonize su damla kaplayın. </li><li> Böylece karın kısmından baş ve toraks kapalı nicking, toraks ve abdomen arasındaki eklem tam sivrisinek karkas kesilirken. </li><li> Sivrisinek kimlik numarası bilgilerini ve uygun gövde bölümünde (; kafa ve göğüs bölümü için &quot;ht&quot; mid-gut bölüm için örnek: &quot;m&quot;) belirtmek için işaretlenmiş olan prelabeled temiz otoklava 1,5 ml mikrosantrifüj tüp içine her disseke sivrisinek bölüm aktarın. </li><li> Parafilm atın ve dikkatlice sonraki sivrisinek işlemeden önce% 70 alkol nemlendirilmiş Kimwipe ile mikroskop sahne silin. </li></ol> 2. Sivrisinek örneklerden DNA Ekstraksiyonu <ol><li> Örnek tüpü ve bir un içine daldırılmış sivrisinek bölümüne öğütmek için kullanıma pipet içine deiyonize su Pipet 20 uliform süspansiyon. </li><li> Otoklavlanmış 1X PBS / nazik anlık vorteks tarafından numune Homojenat ve karıştırmak için 1% saponin çözeltisinden 100 ul ekleyin. </li><li> 20 dakika oda sıcaklığında inkübe edin. </li><li> 2 dakika boyunca 20.000 xg&#39;de santrifüjleyin ve supernatant atın. </li><li> 100 ul 1X PBS içinde yeniden süspanse pelet. </li><li> 2 dakika boyunca 20.000 xg&#39;de tekrar santrifüj ve supernatant atın. </li><li> Yumuşak vorteks (5 sn), 75 ul steril deiyonize su içinde tekrar süspansiyon pelet ve deiyonize su içinde% 20 w / v Chelex-100 reçine süspansiyon 25 ul ile. </li><li> Bunsen beki de Alevli steril 23G derialtı iğne kullanılarak örnek tüpünün kapağını Pierce delik. </li><li> 10 dakika boyunca kayan raf üzerindeki su banyosu içinde kaynatılır örnek süspansiyon (veya ısıtma bloğu). </li><li> PCR uygulamalarında şablon olarak kullanmak için prelabeled saklama kabı içine 1 dk ve transfer eden DNA çözümü için, 20,000 x g&#39;de santrifüjleyin. </li></ol> 3. Plasmodium falciparum genotipleme ve Anofelspp. Moleküler Tanımlanması <ol><li> Anofel türleri 10 belirlemek ve genotip P., DNA kalitesi 11 kontrol etmek için 25 ul PCR reaksiyonlarında Chelex DNA ekstraktı 2.5 ul ekleyin falciparum 12 orta bağırsak ve tükürük bezi enfeksiyonları. </li><li> Özellikle P. için Amplikon verimi maksimize etmek için falciparum algılama, PCR in (BSA), 1.5x bovin serum albümin yer alır. Aşağıdaki reaksiyon bileşim tavsiye edilmektedir: (2.5 ul şablon, 0.25 uM primerler, 1.5 mM magnezyum klorid, 200 uM dNTP, 1X PCR Tamponu, 1.5x BSA ve 1.0U Taq DNA polimeraz, 25 ul hacim içinde). </li><li> Özü DNA kalite kontrol etmek için, eklembacaklı mitokondriyal nikotinamid adenin dinükleotid dehidrogenaz (NADH) altbirim 4 (ND4) gen (primerleri ND4FW [5&#39;-GTD YAT TTA TGA TTR SKK AA-3 &#39;] ve ND4RV [5&#39; bölgesi amplifiye -CTT ÇGD CTT CCW ADW CGT TC-3 &#39;]; ürünün 400bp) 11,13 bekliyoruz. </li><li> Anofel gambia ayırt etmeke sl. türler (An. gambiae ss ve An. arabiensis sadece), intergenik boşluk (IGS) bölgesinde, (SNP sınırdaş bölgede yükseltmek primerler BM [5&#39;-GTGTGCCCCTTCCTCGATGT-3 &#39;], GA [5&#39;-CTGGTTTGGTCGGCACGTTT- 3 &#39;], AR [5&#39;-AAGTGTCCTTCTCCATCCTA-3&#39;]; ürün 390bp bir gambiae ss, 315bp bir arabiensis) 10,11 beklenmektedir. </li><li> Yazarak P. için falciparum antifolate ilaç direnci polimorfizmleri, amino asit, kodonlar 108, 51, 59, parazit dihidrofolat redüktaz (DHFR) geni içinde 16 ve 164 komşu bölgeyi büyütmek için iç içe PCR işlemi gerçekleştirmek: Birincil yuvarlak primerler: M1 [5&#39;-TTTATGATGGAACAAGTCTGC-3 &#39;] ve M5 [5&#39;-AGTATATACATCGCTAACAGA-3&#39;] İkincil yuvarlak primerler: M3 [5&#39;-TTTATGATGGAACAAGTCTGCGACGTT-3 &#39;] F / ile [5&#39;-AAATTCTTGATAAACAACGGAAACCTTTTA-3&#39;] Veya F [5&#39;-GAAATGTAATTCCCTAGATATGGAATATT-3] M4 [5&#39;-TTAATTTCCCAAGTAAAACTATTAGAGCTTC-3 &#39;]). Ayrıca amino asit kodon 436, 437, 540, 581 bir içeren bölge amplifiyeP. nd 613 falciparum Dihidropteroat sentaz (DHPS) geni: Birincil yuvarlak primerler R2 [5&#39;-AACCTAAACGTGCTGTTCAA-3 &#39;] R / [5&#39;-AATTGTGTGATTTGTCCACAA3&#39;]; İkincil yuvarlak primerler J: [5&#39;-&#39;TGCTAGTGTTATAGATATAGGTGGAGAAAGC-3&#39;] K / [5&#39;-CTATAACGAGGTATTGCATTTATTGCAAGAA-3 &#39;] ile Veya K: K / [5&#39;-TGCTAGTGTTATAGATATAGGATGAGCATC-3 &#39;], Veya L [5&#39;-ATAGGATACTATTTGATATTGATATTGGACCAGGATTCG-3 &#39;] L / [5&#39;-5TATTACAACATTTTGATCATTCGCGCAACCGG-3&#39;] 12. </li><li> Üreticinin talimatları izleyerek 30 ul reaksiyon allel-spesifik restriksiyon enzim sindirim, Konu 4 ul Amplikon antifolate ilaç direnci ile ilişkili polimorfizmleri 12 algılamak için. </li><li> Konu 5 elektroforez etidyum bromür ile boyalı% 2&#39;lik agaroz jel (100 - 120V) ve her kulvar için PCR amplikon (veya kısıtlama sindirmek 15 ul) ul ve UV transillüminasyon altında bantları görselleştirmek. </li></ol>

<ol>
	<li>Bonnet, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+antimalarial+treatment+in+Guinea:+in+vivo+study+of+two+artemisinin+combination+therapies+in+Dabola+and+molecular+markers+of+resistance+to+sulphadoxine-pyrimethamine+in+N'Zerekore.">Efficacy of antimalarial treatment in Guinea: in vivo study of two artemisinin combination therapies in Dabola and molecular markers of resistance to sulphadoxine-pyrimethamine in N'Zerekore.</a> Malar. J. 6, 54 (2007).</li><li>Nsimba, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulphadoxine/pyrimethamine+versus+amodiaquine+for+treating+uncomplicated+childhood+malaria+in+Gabon:+a+randomized+trial+to+guide+national+policy.">Sulphadoxine/pyrimethamine versus amodiaquine for treating uncomplicated childhood malaria in Gabon: a randomized trial to guide national policy.</a> Malar. J. 7, 31 (2008).</li><li>Oyewole, I. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behaviour+and+population+dynamics+of+the+major+anopheline+vectors+in+a+malaria+endemic+area+in+southern+Nigeria.">Behaviour and population dynamics of the major anopheline vectors in a malaria endemic area in southern Nigeria.</a> J. Vector Borne Dis. 44, 56-64 (2007).</li><li>Harris, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+proportion+of+asymptomatic+Plasmodium+infections+with+low+and+sub-microscopic+parasite+densities+in+the+low+transmission+setting+of+Temotu+Province,+Solomon+Islands:+challenges+for+malaria+diagnostics+in+an+elimination+setting.">A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting.</a> Malar. J. . 9, 254 (2010).</li><li>Ouedraogo, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substantial+contribution+of+submicroscopical+Plasmodium+falciparum+gametocyte+carriage+to+the+infectious+reservoir+in+an+area+of+seasonal+transmission.">Substantial contribution of submicroscopical Plasmodium falciparum gametocyte carriage to the infectious reservoir in an area of seasonal transmission.</a> PLoS ONE. 4, e8410 (2009).</li><li>Birx, D., de Souza, M., Nkengasong, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+challenges+in+the+scaling+up+of+HIV,+TB,+and+malaria+programs:+The+interaction+of+health+and+laboratory+systems,+clinical+research,+and+service+delivery.">Laboratory challenges in the scaling up of HIV, TB, and malaria programs: The interaction of health and laboratory systems, clinical research, and service delivery.</a> Am. J. Clin. Pathol. 131, 849-851 (2009).</li><li>Ishengoma, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+laboratories+in+the+Tanga+region+of+Tanzania:+the+quality+of+diagnostic+services+for+malaria+and+other+communicable+diseases.">Health laboratories in the Tanga region of Tanzania: the quality of diagnostic services for malaria and other communicable diseases.</a> Ann. Trop. Med. Parasitol. 103, 441-453 (2009).</li><li>Alonso, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+research+agenda+for+malaria+eradication:+vector+control.">A research agenda for malaria eradication: vector control.</a> PLoS Med. 8, e1000401 (2011).</li><li>Grimberg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+non-organic+procedure+for+the+isolation+of+genomic+DNA+from+blood.">A simple and efficient non-organic procedure for the isolation of genomic DNA from blood.</a> Nucleic Acids Res. 17, 8390 (1989).</li><li>Scott, J. A., Brogdon, W. G., Collins, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indentification+of+single+specimens+of+the+Anopheles+gambiae+complex+by+polymerase+chain+reaction.">Indentification of single specimens of the Anopheles gambiae complex by polymerase chain reaction.</a> American Journal of Tropical Medicine and Hygiene. 49, 520-529 (1993).</li><li>Kent, R. J., Norris, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mammalian+blood+meals+in+mosquitoes+by+a+multiplexed+polymerase+chain+reaction+targeting+cytochrome+B.">Identification of mammalian blood meals in mosquitoes by a multiplexed polymerase chain reaction targeting cytochrome B.</a> Am. J. Trop. Med. Hyg. 73, 336-342 (2005).</li><li>Duraisingh, M. T., Curtis, J., Warhurst, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum:+detection+of+polymorphisms+in+the+dihydrofolate+reductase+and+dihydropteroate+synthetase+genes+by+PCR+and+restriction+digestion.">Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion.</a> Exp. Parasitol. 89, 1-8 (1998).</li><li>Gorrochotegui-Escalante, N., Munoz, M. L., Fernandez-Salas, I., Beaty, B. J., Black, W. C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+isolation+by+distance+among+Aedes+aegypti+populations+along+the+northeastern+coast+of+Mexico.">Genetic isolation by distance among Aedes aegypti populations along the northeastern coast of Mexico.</a> Am. J. Trop. Med. Hyg. 62, 200-209 (2000).</li><li>Kreader, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relief+of+amplification+inhibition+in+PCR+with+bovine+serum+albumin+or+T4+gene+32+protein.">Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein.</a> Appl. Environ. Microbiol. 62, 1102-1106 (1996).</li><li>Norris, D. E., Shurtleff, A. C., Toure, Y. T., Lanzaro, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsatellite+DNA+polymorphism+and+heterozygosity+among+field+and+laboratory+populations+of+Anopheles+gambiae+ss+(Diptera+Culicidae).">Microsatellite DNA polymorphism and heterozygosity among field and laboratory populations of Anopheles gambiae ss (Diptera Culicidae).</a> J. Med. Entomol. 38, 336-340 (2001).</li><li>Schriefer, M. E., Sacci, J. B., Wirtz, R. A., Azad, A. F. <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+polymerase+chain+reaction-amplified+malarial+DNA+in+infected+blood+and+individual+mosquitoes.">Detection of polymerase chain reaction-amplified malarial DNA in infected blood and individual mosquitoes.</a> Exp. Parasitol. 73 (91), 311-316 (1991).</li><li>Al-Soud, W. A., Radstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+PCR-inhibitory+components+in+blood+cells.">Purification and characterization of PCR-inhibitory components in blood cells.</a> J. Clin. Microbiol. 39, 485-493 (2001).</li><li>Hyun, C., Filippich, L. J., Hughes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibitory+effect+of+pentobarbitone+on+reverse+transcription-PCR.">The inhibitory effect of pentobarbitone on reverse transcription-PCR.</a> J. Biochem. Biophys. Methods. 62, 63-68 (2005).</li></ol>

Çıkar çatışması ilan etti.

Burada sunulan basitleştirilmiş Chelex yöntemi kalitesi Anopheles spp ve P. çıkarılması sağlar farklı PCR uygulamaları mükellef sivrisinek örneklerden falciparum DNA. Bu teknik, ilaca dirençli P. moleküler sıtma vektör sivrisinek tespiti ve gözetim için istihdam edilebilir ulusal sıtma kontrol programları için sivrisinekler falciparum allel. Basitleştirilmiş Chelex tekniğin avantajları basitlik, daha az reaktif ve dolayısıyla maliyet, güvenlik ve bu yöntemi 10 salting out (2 saat 47 dakika ve bir gecede adım, Tablo 1) gibi standart protokolleri daha kısa işlem süresi (37 dakika) bulunmaktadır. Söz konusu avantajlar ve minimal reaktif gereksinimleri (3 reaktifler) mevcut standart protokol (10 reaktifleri, Tablo 1) 11,15 karşılaştırıldığında, sabit reaktif endemik ülke laboratuvarları basitleştirilmiş Chelex protokolü özellikle kolay haletedarik zinciri çoğu kez sağlamak zordur. Yöntemin özel bir sınırlama standart protokol gibi, aynı zamanda sivrisinek zar 16 meydana geldiği bilinmektedir PCR inhibitörleri tabi olmasıdır. Bununla birlikte, bu kolayca deneylerinde BSA dahil edilmesi ile rahatlatılır. BSA de başarıyla diğer PCR uygulamaları 17,18 inhibitörleri karşı bir amplifikasyon artırıcı olarak istihdam edilmiştir.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Chelex-100, 50-100 kuru örgü </td><td> BioRad </td><td> # 143-2832 </td><td></td></tr><tr><td> Saponin </td><td> SIGMA </td><td> 142-7425 </td><td></td></tr><tr><td> BSA </td><td> New England Biolabs </td><td> B9001S </td><td></td></tr></tbody></table>

a simple chelex protocol for dna extraction from anopheles spp.

Endemik ülkede giderek kontrol programlarının 1,2,3,4,5 ayrılmaz bir parçası olarak, verimli yazarak, tanımlama ve sıtma parazitleri ve vektör sivrisineklere karşı gözetim için moleküler araçlar benimsiyor. Işlemleri bu doğru yaklaşımların sürdürülebilir kurulması için sıtma ve hasis reaktif ve ekipman ihtiyaçları ile eleme çabaları, basit ve ekonomik yöntem, güçlendirmek için araştırma 6,7,8 gereklidir. Burada alanda toplanan sivrisinek örneklerden sıtma parazit ve vektör DNA ayıklamak için basit bir Chelex tabanlı tekniğinin sunulması. Biz morfolojik 72 Anofel gambiae sl belirlenmiştir. Piretrum sprey Macha de Sıtma Enstitüsü 2.000 km 2 civarı içinde evlerin odalarında uyku yakalar tarafından yakalanan 156 sivrisinek. Diseksiyonu 72 Anofel gam için karından baş ve toraks ayırmak için sonrabiae sl. sivrisinekler, iki ayrı bölümden 1.5 mL mikrosantrifüj tüpü içine yerleştirilir ve deiyonize su içinde 20 ul batık edildi. Steril bir pipet ucu kullanarak, her bölüm ayrı ayrı sivrisinek deiyonize su içinde homojen bir süspansiyon ile homojenize edildi. 10 ul başka bir ayrı otoklavlanmış 1.5 ml tüpüne nakledilir iken her bir sivrisinek bölümünden sonraki Homojenat, 10 ul muhafaza edildi. Ayrı alikotları ekstraksiyon protokolü 9,10 salting out basitleştirilmiş Chelex veya standart ya tarafından DNA ekstraksiyonu tabi tutuldu. Bu DNA ekstraksiyon 9 sırasında protein çökeltme adımı için zararlı organik çözücüler (örneğin, fenol ve kloroform gibi) yerine yüksek tuz konsantrasyonları kullanır çünkü dışarı tuzlama protokol olarak adlandırılan ve yaygın olarak kullanılmaktadır. Özler eklembacaklı mitokondriyal nikotinamid adenin dinükleotid dehidrojenaz hedefleme primerler kullanılarak PCR amplifikasyonu için şablon olarak kullanıldıKaraburun (NADH) altbirim 4 gen (ND4) Plasmodium falciparum enfeksiyonu 12 yazmak için DNA kalitesi 11, Anofel gambiae türler 10 tespiti için PCR ve nested PCR kontrol etmek. DNA kalitesi (ND4) kullanarak Karşılaştırma PCR kurulan dışarı tuzlama protokolü göreli Chelex yaklaşım için% 93 duyarlılık ve% 82 özgüllük gösterdi. Duyarlılık ve özgüllük Sorumlu değerleri P. için sırasıyla, türler tanımlama PCR ve% 92 ve% 80 ile, sırasıyla,% 100 ve% 78 idi falciparum tespiti PCR. Chelex veya düzenli her üç PCR uygulamalarında protokolü üzerinden tuzlama ile Amplikon sinyal vererek örneklerin oranları arasında anlamlı farklılık saptanmadı. Chelex yaklaşım üç basit reaktifler ve tamamlamak için 37 dakika gerekli iken bir gecede adım dahil, 10 farklı reaktifler ve 2 saat ve 47 dk &#39;işlem süresi gerektirdiği protokol üzerinde tuzlama. Bizim sonuçlarımız th gösterChelex yöntem de mevcut tuzlama dışarı çıkarma ile karşılaştırılabilir ve sabit bir reaktif tedarik zinciri çoğu kez muhafaza edilmesi güçtür kaynak sınırlı olduğu ortam içinde basit ve sürdürülebilir bir yaklaşım olarak ikame edilebilir.

Sivrisinek DNA ekstraktı kalitesi için PCR (Şekil 1), Anofel arabiensis moleküler tanımlama (Şekil 2) ve P. sonuçları örnekleri basitleştirilmiş Chelex yöntem çok daha az adımda (Tablo 1) rağmen, protokol 10 salting out standart benzer sonuçlar verdiğini falciparum algılama (Şekil 3) göstermektedir. Ilgili özler karşılaştırılabilir DNA kalitesi sayesinde şaşırtıcı olmadığını An ile ilgili örnek pozitiflik oranları. gambiae türler gibi parazit enfeksiyon oranları McNemar ki-kare testi (Şekil 3) göre istatistiksel olarak farklı değildi. Duyarlılık (%) olarak hesaplanmıştır TP / (TP + FN) TP gerçek pozitifler gösterir ve FN yanlış negatifler gösterir * 100,. Özgünlük (%) TN / olarak saptandı (TN + FP) TN gerçek negatifler gösterir ve FP yanlış pozitiflerin gösterir * 100,. DNA kalitesi (ND4) PCR kurulmuş olarak protokolü üzerinden tuzlama altın standardına göre Chelex yaklaşım için% 93 duyarlılık ve% 82 özgüllük gösterdi. Duyarlılık ve özgüllük Sorumlu değerleri P. için sırasıyla, türler tanımlama PCR ve% 92 ve% 80 ile, sırasıyla,% 100 ve% 78 idi falciparum tespiti PCR. Reaksiyon karışımları BSA ek protokolü üzerinden tuzlama basitleştirilmiş Chelex ve düzenli hem nedeniyle PCR inhibitörleri 14 dindirilmesi için PCR pozitif genel bir artış (Şekil 3) sonuçlandı. Ancak bu artış Chelex özler (p = 0.039) üzerinde DNA kalitesi (ND4 PCR) haricinde istatistiksel olarak anlamlı değildi. P. üzerinde Allel özel sınırlama enzimi özümlemesi falciparum DHFR (ya da diğer hedef gen) Amplikon ilaç direnci allelleri (; tükürük bezi veriler gösterilmemiştir Şekil 4) için orta-bağırsak ve tükürük bezi sıtma enfeksiyonu genotipleme sağlar. <table boorder = &quot;1&quot;&gt; <tbody><tr><td colspan="2"> Basit Chelex Prosedürü </td><td colspan="2"> Standart Prosedür üzerinden Tuzlama </td></tr><tr><td> Adımlar </td><td> Reaktifler </td><td> Adımlar </td><td> Reaktifler </td></tr><tr><td><ol><li> 1.5 ml mikrofuge&#39;de tüp numune ekleyin ve 1X PBS /% 1 Saponin solüsyonu (2 dak) 100 ul içerisinde homojenize. </li><li> 20 dakika için oda sıcaklığında bırakın. </li><li> 2 dakika boyunca 20.000 xg&#39;de Spin ve supernatant atın. </li><li> 1X PBS 100 ul ekleyin ve anlık (3 sn) vorteks tarafından yavaşça karıştırın. </li><li> 2 dakika boyunca 20.000 xg&#39;de Spin ve supernatant atın. </li><li> Deiyonize su ve 75 ul steril deiyonize su içinde% 20 w / v Chelex 25 ul ekle. </li><li> 10 dakika sıcak, steril 23G derialtı iğne kaynatın ve tüp içeriğini kullanarak örnek tüpünün kapağını Pierce delik. </li><li> 2 at mikrofuge&#39;de tüp Spin1 dakika için 0,000 xg. </li><li> Kullanılıncaya kadar -20 ° C (25 ul PCR reaksiyonunda 2.5 ul) steril prelabeled flakon ve mağaza DNA çözeltisi içine supernatant aktarın. </li></ol></td><td> PBS Saponin Chelex-100 boncuk </td><td><ol><li> 1.5 ml mikrofuge&#39;de tüp numune ekleyin ve 1% DEPC (2 dak) 100 ul Bender tampon * içerisinde homojenize. </li><li> 1 saat boyunca 65 ° C&#39;de inkübe edin. </li><li> 15 ul soğuk 8 M potasyum asetat ekleyin. Yavaşça karıştırın ve 45 dakika buz üzerinde inkübe edin. </li><li> Yeni bir 1.5 ml tüp supernatant 10 dk ve transferi için 20.000 xg&#39;de mikrofuge&#39;de tüpler örnek Spin. </li><li> % 100 etanol 250 ul ekleyin ve tersini tüp ile iyice karıştırın. </li><li> 5 dakika boyunca oda sıcaklığında numune inkübe edin. </li><li> 15 dakika boyunca 20.000 xg&#39;de örnekleri Spin. </li><li> Aspire ve (30 m tüp pelet tamamen kurumasını bırakarak, supernatant atın) içinde. </li><li> 4 gecede 0,1 X SSC + RNAse (10 ug / ml) 20 ul içinde süspanse pelet ° C. </li><li> Kullanıma kadar -20 ° C&#39;de 80 ul DEPC su ve mağaza ekleyin. </li></ol></td><td> Diethylpyrocarbonate (DEPC) * Bender Tampon: 0.1 M NaCl 0.2 M Sakkaroz 0.1 M Tris-HCL 0.05 M EDTA, pH 9.1 &#39; DEPC su içinde% 0.5 SDS 8 M Potasyum asetat Etanol 0,1 X Tuzlu sodyum sitrat (SSC) tampon RNAse (10 ug / ml) </td></tr><tr><td colspan="2"> TOPLAM SÜRE: 37 dk </td><td colspan="2"> TOPLAM SÜRE: 2 saat 47 dk, artı gecede </td></tr></tbody></table> Tablo 1. Basit Chelex protokolü için reaktifler ve zaman gereksinimleri adım karşılaştırma adım ve standard sivrisinek örneklerinden DNA ekstraksiyonu için yöntemi tuzlama. <img alt="Şekil 1" src="/files/ftp_upload/3281/3281fig1.jpg" /> Şekil 1. ND4 mitokondriyal PCR basitleştirilmiş Chelex &quot;C&quot; DNA kalitesinin karşılaştırılması ve Anofel arabiensis alan numuneler üzerinde &quot;M&quot; özler dışarı tuzlama standart. NC, negatif kontrol; M1 ve C1, M2 ve C2, M3 ve C3, ve M4 ve C4 üzerinden tuzlama ve aynı bir araba Chelex özler eşleştirilir. arabiensis sivrisinek örnekleri. L, 100 bp DNA ladder. <img alt="Şekil 2," src="/files/ftp_upload/3281/3281fig2.jpg" /> Şekil 2 Anofel arabiensis için Moleküler tanımlama PCR C1 ve M1, C2 ve M2, C3 ve M3, C4 ve M4 &quot;M&quot; salting out ve aynı sivrisinek numuneler için Chelex &quot;C&quot; DNA ekstraktları basitleştirilmiş itibaren ilgili Amplikon temsil eder;.. AR, Anofel arabiensisPozitif kontrol, L, 100 bp DNA ladder. <img alt="Şekil 3" src="/files/ftp_upload/3281/3281fig3.jpg" /> Şekil 3. Chelex ve Anofel arabiensis (AR) 10, artropod NADH dehidrogenaz geninin 4 (ND4) DNA kalitesi testi 11 ve antifolate ilaç direnci P moleküler tespiti için PCR ile, sivrisinek alan örnekleri DNA özü dışarı Tuzlanmaya pozitiflerin Algılama . falciparum DHFR genotipleme 12 (F-M4). Reaksiyon karışımı BSA ile veya olmadan çalıştırabilirsiniz deneyleri için gösterilen sonuçlar. McNemar ki-kare testi DNA pozitif PCR yüzde arasındaki farklılıklar basitleştirilmiş Chelex çıkarılan ve protokolleri tuzlama standart istatistiksel olarak anlamlı olup olmadığını belirlemek için kullanılmıştır. <img alt="Şekil 4," src="/files/ftp_upload/3281/3281fig4.jpg" /> <strong&gt; Şekil 4. genotipleme P. basitleştirilmiş Chelex protokolünün uygulanması ilaç direnci alleller için sivrisinekler falciparum enfeksiyonlar (orta-gut veriler gösterilmemiştir). P. kodon 108 sınırdaş Amplikon ilgili BstN Ben sindirim falciparum DHFR gen 12 sivrisinek örnekleri (; mid-gut veriler gösterilmemiştir M1-M5) in cycloguanil dayanıklı S108T mutantlar gösterir. U, sindirilmemiş 522 bp amplikon; FCR3, laboratuvar standart P. S108T taşıyan falciparum pozitif kontrol klonu; K1, P. falciparum laboratuvar standart cycloguanil duyarlı S108N taşıyan klon negatif kontrol; L, 100 bp DNA ladder.

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

Anopheles spp.'den DNA Ekstraksiyonu için Basit Bir Chelex Protokolü. | İmmünoloji ve Enfeksiyon | Video

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Sivrisinek örneklerden kalitesi sıtma parazit ve vektör DNA ayıklamak için bir hızlı ve ekonomik bir şekilde açıklanmıştır. Basit bir yöntem sivrisinek orta bağırsak ve tükürük bezi aşamalarında sıtma parazitlerinin genotipleme yanı sıra moleküler tanımlanması, Chelex reçine çelatlama özelliklerine etkinleştirir sermaye<em&gt; Anofel</em&gt; PCR ile türlerin kardeş.

DNA ekstraksiyonu için A Simple Chelex Protokolü<em&gt; Anofel</em&gt; Spp.

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

a-simple-chelex-protocol-for-dna-extraction-from-anopheles-spp

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Immunology and Infection

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Genetics

DNA Structure and Function

SCIENTISTS IN ACTION

54.7K Görüntüleme.  Malaria Institute at Macha. Sivrisinek örneklerden kalitesi sıtma parazit ve vektör DNA ayıklamak için bir hızlı ve ekonomik bir şekilde açıklanmıştır. Basit bir yöntem sivrisinek orta bağırsak ve tükürük bezi aşamalarında sıtma parazitlerinin genotipleme yanı sıra moleküler tanımlanması, Chelex reçine çelatlama özelliklerine etkinleştirir sermaye Anofel PCR ile türlerin kardeş.

Video: 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.

Yellow Fever Sivrisinek Hemocytes Toplama ve Boyama için Protokol 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 ...

İmmünoloji ve Enfeksiyon

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 Teknoloji: Yumurta Sarısı Tavuk Antikorlar çıkarımı Polietilen Glikol (PEG) Yağış

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.

Kök-ur Nematod ile Yüksek ve Düşük Throughput Ekranlar 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.

Trombosit-aracılı kesekleşme için Basit Protokolü<em&gt; Plasmodium falciparum</emBir kaynak Poor Setting&gt; ile enfekte Eritrosit

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

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Toplam Protein Ekstraksiyon ve 2-D Jel Elektroforez Yöntemleri<em&gt; Burkholderia</em&gt; Türler

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatit C Virüsü Çoğaltma Analizi için Protokol

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

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.

İnfiltratif Lenfositlerinin Analiz Olmayan enzimatik ayırmak Taze İnsan Dokusuna Basit ve Hızlı Protokolü

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

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

İnsan Palatine Bademcikler B ve T Lenfosit için Verimli İzolasyon Protokolü

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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.

Basit ve Etkin Yaklaşım Mutant Vaccinia Virüs Vektörler Construct

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

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.