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Liquid biopsy (LB) nigitekerezo kigenda cyamamara vuba murwego rwibinyabuzima.Igitekerezo gishingiye ahanini ku gutahura ibice bya ADN idasanzwe (ccfDNA), bisohoka cyane nk'uduce duto nyuma y'urupfu rw'uturemangingo mu ngingo zitandukanye.Umubare muto wibi bice ukomoka mubice byamahanga (mumahanga) cyangwa ibinyabuzima.Mubikorwa byubu, twakoresheje iki gitekerezo kuri mussel, ubwoko bwa sentinel buzwiho ubushobozi bwo kuyungurura amazi yo mu nyanja.Dukoresha ubushobozi bwa mussele bwo gukora nkayunguruzo karemano kugirango dufate ibice bya ADN bidukikije biva ahantu hatandukanye kugirango dutange amakuru kubyerekeranye nibinyabuzima bitandukanye by urusobe rwibinyabuzima byo ku nyanja.Ibisubizo byacu byerekana ko mussel hemolymph irimo ibice bya ADN bitandukanye cyane mubunini, kuva kuri 1 kugeza kuri 5 kb.Urukurikirane rw'amasasu rwerekanye ko umubare munini w'uduce twa ADN dukomoka kuri mikorobe y'amahanga.Muri byo, twasanze ibice bya ADN biva muri bagiteri, archaea, na virusi, harimo na virusi zizwiho kwanduza abantu benshi bakunze kuboneka mu bidukikije byo ku nyanja.Mu gusoza, ubushakashatsi bwacu bugaragaza ko igitekerezo cya LB gikoreshwa kuri mussele kigaragaza isoko ikungahaye ariko itaracukumburwa yubumenyi bujyanye na mikorobe itandukanye yibinyabuzima byo ku nyanja.
Ingaruka z’imihindagurikire y’ikirere (CC) ku binyabuzima bitandukanye by’ibinyabuzima byo mu nyanja ni agace k’ubushakashatsi kiyongera cyane.Ubushyuhe bukabije ku isi ntibutera guhangayika gusa, ahubwo binasunika imipaka y’ubwihindurize bw’ubushyuhe bw’imiterere y’ibinyabuzima byo mu nyanja, bigira ingaruka ku moko y’ibinyabuzima byinshi, bigatuma bashakisha ahantu heza [1, 2].Usibye kugira ingaruka ku binyabuzima bitandukanye bya metazoans, CC ihungabanya uburinganire bworoshye bwimikoranire ya mikorobe.Iyi mikorobe ya dysbacteriose ibangamiye cyane urusobe rw’ibinyabuzima byo mu nyanja kuko ituma ibinyabuzima byo mu nyanja byibasirwa na virusi zandura [3, 4].Bikekwa ko SS igira uruhare runini mu rupfu rw’abantu benshi, kikaba ari ikibazo gikomeye ku micungire y’ibinyabuzima byo mu nyanja ku isi [5, 6].Iki nikibazo gikomeye ukurikije ingaruka zubukungu, ibidukikije nimirire yibinyabuzima byinshi byo mu nyanja.Ibi ni ukuri cyane cyane ku bahanganye baba mu turere twa polar, aho ingaruka za CK zihita kandi zikomeye [6, 7].Mubyukuri, bivalves nka Mytilus spp.zikoreshwa cyane mugukurikirana ingaruka za CC kubinyabuzima byo mu nyanja.Ntabwo bitangaje, umubare munini ugereranije na biomarkers wateguwe kugirango ukurikirane ubuzima bwabo, akenshi ukoresheje uburyo bubiri burimo biomarkers ikora ishingiye kubikorwa byimikorere cyangwa imikorere ya selile nkibikorwa bya selile nibikorwa bya fagocyitike [8].Ubu buryo kandi bukubiyemo gupima ubunini bwibipimo byerekana umuvuduko ukusanyiriza mu ngingo zoroheje nyuma yo kwinjiza amazi menshi yo mu nyanja.Nyamara, ubushobozi bwo kuyungurura cyane hamwe na sisitemu yo gufungura igice cya kabiri cya bivalves itanga amahirwe yo guteza imbere biomarkers nshya ya hemolymph ikoresheje igitekerezo cya biopsy yamazi (LB), uburyo bworoshye kandi bworoshye bwo gucunga abarwayi.icyitegererezo cy'amaraso [9, 10].Nubwo ubwoko butandukanye bwa molekile zuzunguruka zishobora kuboneka muri LB yumuntu, iki gitekerezo gishingiye cyane cyane kubisesengura rya ADN ikurikirana ryikwirakwizwa rya ADN idasanzwe (ccfDNA) muri plasma.Mubyukuri, kuba ADN ikwirakwizwa muri plasma yabantu byamenyekanye kuva mu kinyejana cya 20 rwagati [11], ariko mu myaka yashize ni bwo haje uburyo bwo gutondekanya ibintu byinshi byatumye hasuzumwa ivuriro rishingiye kuri ccfDNA.Kuba hari ibice bya ADN bizenguruka biterwa no kurekura byimazeyo ADN ya genomic (nucleaire na mitochondrial) nyuma y'urupfu rw'utugari. Ku bantu bafite ubuzima bwiza, ubusanzwe ccfDNA iba mike (<10 ng / mL) ariko irashobora kwiyongera inshuro 5-10 kubarwayi barwaye indwara zitandukanye cyangwa bahuye nibibazo, bikaviramo kwangirika kwinyama. Ku bantu bafite ubuzima bwiza, ubusanzwe ccfDNA iba mike (<10 ng / mL) ariko irashobora kwiyongera inshuro 5-10 kubarwayi barwaye indwara zitandukanye cyangwa bahuye nibibazo, bikaviramo kwangirika kwinyama. У здоровых людей маран вккДНК в назме низкая (<10 нг / ھى) Ku bantu bafite ubuzima bwiza, ubusanzwe bwa cccDNA iba mike (<10 ng / mL), ariko irashobora kwiyongera inshuro 5-10 kubarwayi bafite indwara zitandukanye cyangwa bahangayitse biganisha ku kwangirika kwinyama.在 健康 个体 中 c ccfDNA 的 浓度 通常 较低 （<10 ng / mL） ， 但 在 患有 各种 或 承受 -10 -10 -10 -10 -10 -10 5-10 倍 ， 从而 导致 组织 损伤。。在 健康 个体 中 c ccfdna 的 浓度 较 低 （（<10 ng / ml） 但 在 各 种 病理 -10 -10 5-10 倍 ， 从而 组织 -10 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤Концентрации ccfDNA обохно низкие (<10 нг / كان) у здоровых людей, но могут быть увеличены в 5-10 раз у удов с различными пидологиями или чисом, что при имитит к повреждению тканей. Ubushuhe bwa ccfDNA mubusanzwe buri hasi (<10 ng / ml) kubantu bafite ubuzima bwiza, ariko birashobora kwiyongera inshuro 5-10 kubarwayi bafite indwara zitandukanye cyangwa imihangayiko, bikaviramo kwangirika kwinyama.Ingano y'ibice bya ccfDNA iratandukanye cyane, ariko mubisanzwe iri hagati ya 150 na 200 bp.[12].Isesengura ryakozwe na ccfDNA yonyine, ni ukuvuga, ccfDNA iva mu ngirabuzimafatizo zisanzwe cyangwa zahinduwe, irashobora gukoreshwa mu kumenya impinduka zishingiye ku ngirabuzima fatizo na epigenetike ziri muri genome ya kirimbuzi na / cyangwa mitochondrial, bityo bigafasha abaganga guhitamo imiti yihariye igamije kuvura [13].Ariko, ccfDNA irashobora kuboneka mumasoko yamahanga nka ccfDNA ivuye mumyanya myibarukiro mugihe utwite cyangwa mubice byatewe [14,15,16,17].ccfDNA kandi nisoko yingenzi yamakuru yo kumenya ko habaho aside nucleique ya agent yanduye (mumahanga), ituma hatabaho kwandura indwara zanduye zitagaragajwe numuco wamaraso, ukirinda biopsy yibasira ingirangingo zanduye [18].Ubushakashatsi buherutse gukorwa bwerekanye rwose ko amaraso y’abantu arimo amakuru menshi ashobora gukoreshwa mu kumenya virusi na bagiteri, kandi ko hafi 1% ya ccfDNA iboneka muri plasma y’abantu ikomoka mu mahanga [19].Ubu bushakashatsi bwerekana ko ibinyabuzima bitandukanye bya mikorobe ikwirakwiza ibinyabuzima bishobora gusuzumwa hifashishijwe isesengura rya ccfDNA.Ariko, kugeza vuba aha, iki gitekerezo cyakoreshwaga gusa mubantu kandi, ku rugero ruto, mu zindi nyababyeyi [20, 21].
Muri iyi nyandiko, dukoresha ubushobozi bwa LB kugirango dusesengure ccfDNA ya Aulacomya atra, ubwoko bwamajyepfo bukunze kuboneka mu birwa bya Kerguelen bya subantarctique, itsinda ryibirwa hejuru yikibaya kinini cyashinzwe mu myaka miriyoni 35 ishize.ikirunga.Twifashishije sisitemu yubushakashatsi bwa vitro, twasanze ibice bya ADN mumazi yinyanja bifatwa vuba na mussele hanyuma byinjira mubice bya hemolymph.Urukurikirane rw'amasasu rwerekanye ko mussel hemolymph ccfDNA ikubiyemo ibice bya ADN bikomokaho kandi bitari ubwabyo, harimo na bagiteri ya symbiotic hamwe na ADN ibice biva muri biome bisanzwe bikomoka ku birunga bikonje byo mu nyanja bikonje.Hemolymph ccfDNA ikubiyemo kandi virusi zikurikirana zikomoka kuri virusi zifite intera zitandukanye.Twasanze kandi ibice bya ADN biva mu nyamaswa nyinshi nk'amafi yo mu magufa, anemone yo mu nyanja, algae n'udukoko.Mu gusoza, ubushakashatsi bwacu bwerekana ko igitekerezo cya LB gishobora gukoreshwa neza mubidafite ubuzima bwinyanja kugirango bibyare repertoire ikungahaye mubinyabuzima byo mu nyanja.
Abakuze (mm 55-70 z'uburebure) Mytilus platensis (M. platensis) na Aulacomya atra (A. atra) bakusanyirijwe mu nkombe z’urutare hagati ya Port-au-Ubufaransa (049 ° 21.235 S, 070 ° 13.490 E.).Ibirwa bya Kerguelen mu Kuboza 2018. Abandi bakuze b'ubururu bakuze (Mytilus spp.) Babonetse ku isoko ry'ubucuruzi (PEI Mussel King Inc, Prince Edward Island, Kanada) maze bishyirwa mu kigega cyagenzuwe n'ubushyuhe (4 ° C) kirimo umwuka wa 10-20 L wa 32 ‰.(umunyu winyanja winyanja Reef Crystal, Inyanja ako kanya, Virginia, USA).Kuri buri bushakashatsi, hapimwe uburebure nuburemere bwibishishwa byihariye.
Porotokole ifunguye kubuntu kuriyi gahunda iraboneka kumurongo (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1).Muri make, LB hemolymph yakusanyirijwe mumitsi yabashimusi nkuko byasobanuwe [22].Hemolymph yasobanuwe na centrifugation kuri 1200 × g mu minota 3, ndengakamere yarahagaritswe (-20 ° C) kugeza ikoreshejwe.Mu kwigunga no kweza cfDNA, ingero (1.5-2.0 ml) zashwanyaguwe kandi zitunganywa hifashishijwe ibikoresho bya NucleoSnap cfDNA (Macherey-Nagel, Bethlehen, PA) ukurikije amabwiriza yabakozwe.ccfDNA yabitswe kuri -80 ° C kugeza irindi sesengura.Mu bushakashatsi bumwe na bumwe, ccfDNA yitaruye kandi isukurwa hifashishijwe ibikoresho bya QIAamp ushinzwe iperereza (QIAGEN, Toronto, Ontario, Kanada).ADN yatunganijwe yagereranijwe hifashishijwe ibipimo bisanzwe bya PicoGreen.Gukwirakwiza ibice bya ccfDNA yitaruye byasesenguwe na capillary electrophoresis ikoresheje bioanalyser ya Agilent 2100 (Agilent Technologies Inc., Santa Clara, CA) ikoresheje ibikoresho bya ADN bikabije.Isuzuma ryakozwe hakoreshejwe 1 µl yicyitegererezo cya ccfDNA ukurikije amabwiriza yabakozwe.
Kugirango ukurikirane ibice bya hemolymph ccfDNA, Génome Québec (Montreal, Quebec, Kanada) yateguye amasomero yimbunda akoresheje ibikoresho bya Illumina ADN bivanga ibikoresho bya Illumina MiSeq PE75.Adapter isanzwe (BioO) yakoreshejwe.Amadosiye yamakuru arahari araboneka muri NCBI Urukurikirane Soma Ububiko (SRR8924808 na SRR8924809).Ubwiza bwibanze bwo gusoma bwasuzumwe hifashishijwe FastQC [23].Trimmomatic [24] yakoreshejwe mugukata adapteri no gusoma nabi.Shotgun isoma ifite imitwe yombi yari FLASH yahujwe no gusoma igihe kirekire hamwe byibuze byibuze 20 bp kugirango birinde kudahuza [25]. Gusoma byahujwe byasobanuwe hamwe na BLASTN ukoresheje bivalve ya NCBI ya Taxonomy (e agaciro <1e - 3 na 90% homologiya), kandi guhisha urutonde rwibintu bito byakorwaga hakoreshejwe DUST [26]. Gusoma byahujwe byasobanuwe hamwe na BLASTN ukoresheje bivalve ya NCBI ya Taxonomy (e agaciro <1e - 3 na 90% homologiya), kandi guhisha urutonde rwibintu bito byakorwaga hakoreshejwe DUST [26]. Объединенные ч чия были аннотирован с помощу BLASTN с использованием басы Кных так такомии двустворч чатх моллюсков NCBI (значение e <1e-3 и 90% tutологии) зованием DUST [26]. Ibisomwa byuzuye byasobanuwe hamwe na BLASTN ukoresheje data base ya NCBI bivalve taxonomy (e agaciro <1e-3 na 90% homology), kandi maskike ikurikirana ikurikirana ikoresheje DUST [26].使用 双壳类 NCBI 分类 数据库 （e 值 <1e-3 和 90% 同源 性） 用 BLASTN 注释 合并 的 读数 ， 并 UST DUST [26] 进行 低 复杂 度 序列 的 掩蔽使用 双 壳 类 ncbi 分类 （（（<1e-3 和 90% 同源） 用 用 使用 使用 使用 使用 使用 使用 使用 ivumbi [26] 进行 复杂 度 序列 。。。。 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽Объединенные ч чения были аннотирован с помощу BLASTN с использованием так консомической быгыныных двустворч чатх моллюсков NCBI (значение e <1e-3 и 90% ологجودوسوۋ) пользованием DUST [26]. Ibisomwa byuzuye byasobanuwe hamwe na BLASTN ukoresheje data base ya NCBI bivalve ya taxonomic (e agaciro <1e-3 na 90% homology), kandi maskike ikurikirana ikurikirana ikoresheje DUST [26].Gusoma byagabanijwemo amatsinda abiri: ajyanye na bivalve ikurikiranye (hano bita kwiyitirira) kandi ntaho bihuriye (kutisoma).Amatsinda abiri yakusanyirijwe hamwe akoresheje MEGAHIT kugirango atange amakimbirane [27].Hagati aho, ikwirakwizwa rya tagisi ya microbiome yasomwe yashyizwe mu byiciro hakoreshejwe Kraken2 [28] kandi igaragazwa neza n'imbonerahamwe ya pie ya Krona kuri Galaxy [29, 30].Ibyiza bya kmers byariyemeje kuba kmers-59 duhereye kubushakashatsi bwambere. Kwiyitirira abandi noneho byamenyekanye muguhuza na BLASTN (bivalve data base ya NCBI, e agaciro <1e - 10 na 60% homology) kugirango tumenye bwa nyuma. Kwiyitirira abandi noneho byamenyekanye muguhuza na BLASTN (bivalve data base ya NCBI, e agaciro <1e - 10 na 60% homology) kugirango tumenye bwa nyuma. Затем собственные эштиги были идент фицированы путем сопоставления с BLASTN (Баа нных двустворч матх моллюсков NCBI, значение e <1e-10 и kurдология 60%) для окончательной аннотации. Kwiyitirira noneho byamenyekanye muguhuza na BLASTN (NCBI bivalve base base, e agaciro <1e-10 na 60% homology) kugirango tumenye bwa nyuma.然后 通过 与 BLASTN （双 壳 贝类 NCBI 数据库 ， e 值 <1e-10 和 60% 同源 性） 对齐 来 识别 自身 重叠 群 以 进行 最终 注释然后 通过 与 BLASTN （双 壳 贝类 NCBI 数据库 ， e 值 <1e-10 和 60% Затем были идент Фифицированя собственные шәһәртиги для окончательной аннотации путем сопоставления с BLASTN (babaа чныхх NCBI для двустворч чехх моллюсков, значение e <1e-10 и зогол 60%). Kwiyitirira noneho byamenyekanye kugirango bisobanurwe bwa nyuma bihuza na BLASTN (NCBI bivalve base, e agaciro <1e-10 na 60% homology). Mu buryo bubangikanye, itsinda ridafite itsinda ryanditswemo na BLASTN (nt NCBI base, e agaciro <1e - 10 na 60% homology). Mu buryo bubangikanye, itsinda ridafite itsinda ryanditswemo na BLASTN (nt NCBI base, e agaciro <1e - 10 na 60% homology). Параллельно чужеродные групповые бастиги были аннотирования с помощью BLASTN (Баа нныхх nt NCBI, значение e <1e-10 и курология 60%). Mu buryo bubangikanye, itsinda ry’amahanga ryasobanuwe na BLASTN (Ububiko bwa NT NCBI, e agaciro <1e-10 na 60% homology).平行 地 ， 用 BLASTN （nt NCBI 数据库 ， e 值 <1e-10 和 60% 同源 性） 注释 非 自身 组 重叠 群。平行 地 ， 用 BLASTN （nt NCBI 数据库 ， e 值 <1e-10 和 60% 同源 性） 注释 非 自身 组 重叠 群。 Параллельно карьертиги, не относящиеся к собственной группе, были аннотирован с помощу BLASTN (باسا كاننىخ nt NCBI, значение e <1e-10 и кидология 60%). Mu buryo bubangikanye, amatsinda adaharanira inyungu yasobanuwe na BLASTN (nt NCBI base base, e agaciro <1e-10 na 60% homology). BLASTX nayo yakorewe kumuntu atabigenewe akoresheje ububiko bwa nr na RefSeq protein NCBI (e agaciro <1e - 10 na 60% homology). BLASTX nayo yakorewe kumuntu atabigenewe akoresheje ububiko bwa nr na RefSeq protein NCBI (e agaciro <1e - 10 na 60% homology). BLASTX takже был проведен на несамамостоятельных кстигах с использованием базных белка nr и RefSeq NCBI (значение e <1e-10 и идология 60%). BLASTX nayo yakorewe kubatari bonyine bakoresheje ububiko bwa protein nr na RefSeq NCBI (e agaciro <1e-10 na 60% homology).还 nr 和 RefSeq 蛋白 NCBI 数据库 对 非 自身 重叠 群 进行 了 BLASTX （e 值 <1e-10 和 60% 同源 性）。还 nr 和 RefSeq 蛋白 NCBI 数据库 对 非 自身 重叠 群 进行 了 BLASTX （e 值 <1e-10 和 60% 同源 性）。 BLASTX takже выполняли на несамамостоятельных хезмәттигах с использованием басныны белка nr na RefSeq NCBI (значение e <1e-10 и идология 60%). BLASTX nayo yakorewe kubatari bonyine bakoresheje ububiko bwa protein nr na RefSeq NCBI (e agaciro <1e-10 na 60% homology).Ibidendezi bya BLASTN na BLASTX byo kutiyitirira-bigereranya ibyanyuma (reba dosiye yinyongera).
Intangiriro zikoreshwa kuri PCR ziri kurutonde rwa S1.Taq ADN polymerase (Bio Basic Canada, Markham, ON) yakoreshejwe mugukomeza ingirabuzimafatizo ya ccfDNA.Uburyo bukurikira bwakoreshejwe bwakoreshejwe: gutandukana kuri 95 ° C muminota 3, 95 ° C kumunota 1, gushiraho ubushyuhe bwa annealing kumunota 1, kurambura kuri 72 ° C kumunota 1, inzinguzingo 35, hanyuma amaherezo 72 ° C muminota 10..Ibicuruzwa bya PCR byatandukanijwe na electrophoreis muri geles ya agarose (1.5%) irimo SYBRTM Yizewe ya ADN Yizewe (Invitrogen, Burlington, ON, Kanada) kuri 95 V.
Mussels (Mytilus spp.) Yamenyereye mumazi 500 ya ogisijeni yo mu nyanja (32 PSU) amasaha 24 kuri 4 ° C.ADN ya plasmide irimo insimburangingo ikubiyemo urutonde rwa galectine-7 ya cDNA yumuntu (NCBI yinjira numero L07769) yongewe kuri vial kumurongo wa 190 μg / μl.Mussels yubatswe mubihe bimwe nta ADN yongeyeho niyo yagenzuraga.Ikigega cya gatatu cyo kugenzura cyarimo ADN idafite imitsi.Kugenzura ubwiza bwa ADN mu mazi yo mu nyanja, ingero z’amazi yo mu nyanja (20 μl; inshuro eshatu zisubiramo) zafashwe kuri buri kigega mugihe cyerekanwe.Kugirango ADN ikurikiranwe neza, imitsi ya LB yasaruwe mugihe cyerekanwe kandi isesengurwa na qPCR na ddPCR.Bitewe numunyu mwinshi wamazi yinyanja, alikoti yavomwe mumazi meza ya PCR (1:10) mbere yubushakashatsi bwa PCR.
Digital droplet PCR (ddPCR) yakozwe hakoreshejwe protocole ya BioRad QX200 (Mississauga, Ontario, Kanada).Koresha imiterere yubushyuhe kugirango umenye ubushyuhe bwiza (Imbonerahamwe S1).Ibitonyanga byakozwe hakoreshejwe imashini itanga QX200 (BioRad).ddPCR yakozwe kuburyo bukurikira: 95 ° C kuminota 5, inzinguzingo 50 za 95 ° C kuri 30 s hamwe nubushyuhe bwa annealing kuminota 1 na 72 ° C kuri 30 s, 4 ° C kuminota 5 na 90 ° C muminota 5.Umubare wibitonyanga nibisubizo byiza (umubare wa kopi / µl) wapimwe ukoresheje umusomyi wa QX200 (BioRad).Ingero zifite ibitonyanga bitarenga 10,000.Igenzura nticyakozwe buri gihe ddPCR yakoreshwaga.
qPCR yakozwe hakoreshejwe Rotor-Gene® 3000 (Ubushakashatsi bwa Corbett, Sydney, Ositaraliya) na LGALS7 primers yihariye.PCR zose zuzuye zakozwe muri 20 µl ukoresheje QuantiFast SYBR Green PCR Kit (QIAGEN).qPCR yatangijwe nubushakashatsi bwa min 15 kuri 95 ° C ikurikirwa ninzinguzingo 40 kuri 95 ° C kumasegonda 10 na 60 ° C kumasegonda 60 hamwe no gukusanya amakuru.Gushonga umurongo byakozwe hakoreshejwe ibipimo bikurikirana kuri 95 ° C kuri 5 s, 65 ° C kuri 60 s, na 97 ° C kumpera ya qPCR.Buri qPCR yakorewe muri bitatu, usibye kugenzura ibyitegererezo.
Kubera ko imitsi izwiho umuvuduko mwinshi wo kuyungurura, twabanje gukora iperereza niba ishobora kuyungurura no kugumana ibice bya ADN biboneka mumazi yinyanja.Twari dushishikajwe kandi no kumenya niba ibyo bice byegeranya muri sisitemu ya lymphatike.Twakemuye iki kibazo mubigeragezo dukurikirana amaherezo ya ADN ibora yongewe kuri tanks yubururu.Kugira ngo tworohereze gukurikirana ibice bya ADN, twakoresheje ADN yo mu mahanga (ntabwo yonyine) plasmid irimo gene ya galectine-7.ddPCR ikurikirana ibice bya ADN ya plasmid mumazi yinyanja na mussele.Ibisubizo byacu byerekana ko niba ingano ya ADN mu mazi yo mu nyanja yagumye ihoraho mugihe (kugeza ku minsi 7) mugihe hatabayeho imitsi, noneho imbere ya mussele uru rwego rwarazimye burundu mumasaha 8 (Ishusho 1a, b).Ibice bya ADN idasanzwe byamenyekanye bitarenze iminota 15 mumazi yo mu nda na hemolymph (Ishusho 1c).Ibi bice birashobora kuboneka kugeza amasaha 4 nyuma yo kugaragara.Iki gikorwa cyo kuyungurura kubijyanye na ADN igereranywa nigikorwa cyo kuyungurura za bagiteri na algae [31].Ibisubizo byerekana ko imitsi ishobora gushungura no kwegeranya ADN yo mumahanga mubice byabo byamazi.
Ubwinshi bwa ADN ya plasmide mumazi yinyanja ahari (A) cyangwa idahari (B) ya mussele, bipimwa na ddPCR.Muri A, ibisubizo bigaragazwa nkijanisha, hamwe nimbibi zamasanduku zerekana 75 na 25 kwijana.Ikirangantego cya logarithmic cyerekanwe gitukura, kandi agace gatwikiriye ibara ryerekana 95% intera yicyizere.Muri B, umurongo utukura ugereranya ikigereranyo naho umurongo wubururu ugereranya 95% intera intera yo kwibanda.C Gukusanya ADN ya plasmide muri hemolymph na valvular fluid ya mussele mubihe bitandukanye nyuma yo kongeramo ADN ya plasmide.Ibisubizo bitangwa nka kopi yuzuye yagaragaye / mL (± SE).
Ubukurikira, twasesenguye inkomoko ya ccfDNA mu mitsi yakusanyirijwe mu buriri bwa mussel ku birwa bya Kerguelen, itsinda rya kure ry’ibirwa bifite imbaraga nke za antropogene.Kubwiyi ntego, cccDNA ivuye muri mussel hemolymphs yitaruye kandi isukurwa nuburyo bukoreshwa mugusukura cccDNA yabantu [32, 33].Twabonye ko impuzandengo ya hemolymph ccfDNA yibitseho imitsi iri muri microgrammes nkeya kuri ml hemolymph (reba Imbonerahamwe S2, Amakuru yinyongera).Uru ruhererekane rwinshi ni runini kuruta kubantu bafite ubuzima bwiza (nanogramu nkeya kuri mililitiro), ariko mubihe bidasanzwe, kubarwayi ba kanseri, urwego rwa ccfDNA rushobora kugera kuri microgramu nyinshi kuri mililitiro [34, 35].Isesengura ryubunini bwikwirakwizwa rya hemolymph ccfDNA ryerekanye ko ibyo bice bitandukanye cyane mubunini, kuva kuri 1000 bp kugeza 1000 bp.kugeza 5000 bp (Ishusho 2).Ibisubizo nkibi byabonetse hakoreshejwe QIAamp Investigator Kit ishingiye kuri silika, uburyo bukoreshwa cyane mubumenyi bwubucamanza kugirango bwigunge kandi busukure ADN genomic duhereye kuri ADN nkeya, harimo ccfDNA [36].
Uhagarariye ccfDNA electrophoregram ya mussel hemolymph.Yakuwe hamwe na NucleoSnap Plasma Kit (hejuru) hamwe na QIAamp ADN ishinzwe iperereza.B Umugambi wa Violin werekana ikwirakwizwa rya hemolymph ccfDNA yibanze (± SE) mumitsi.Imirongo y'umukara n'umutuku igereranya median na quartile ya mbere n'iya gatatu.
Hafi ya 1% ya ccfDNA mubantu na primates ifite isoko yamahanga [21, 37].Dufatiye ku gice cya kabiri gifungura uburyo bwo kuzenguruka, amazi yo mu nyanja akungahaye kuri mikorobe, hamwe no gukwirakwiza ingano ya ccfDNA, twavuze ko mussel hemolymph ccfDNA ishobora kuba irimo pisine ikungahaye kandi itandukanye ya ADN ya mikorobe.Kugirango dusuzume iyi hypothesis, twakurikiranye hemolymph ccfDNA yo muri Aulacomya atra ingero yakusanyirijwe mu birwa bya Kerguelen, itanga ibisobanuro birenga miliyoni 10, 97,6% muri byo byatsinze igenzura.Ibisomwa noneho byashyizwe mubyiciro ukurikije ibyigenga kandi bitari ubwabyo ukoresheje ububiko bwa BLASTN na NCBI bivalve (Ishusho S1, Amakuru yinyongera).
Mu bantu, ADN ya kirimbuzi na mitochondial irashobora kurekurwa mu maraso [38].Nyamara, muri ubu bushakashatsi, ntabwo byashobokaga gusobanura mu buryo burambuye ADN ya genomic ADN ya mussele, bitewe nuko genome ya A. atra itigeze ikurikiranwa cyangwa ngo isobanurwe.Ariko, twashoboye kumenya umubare wibice bya ccfDNA dukomokaho dukoresheje isomero rya bivalve (Ishusho S2, Amakuru yinyongera).Twemeje kandi ko hari ibice bya ADN bituruka ku nkomoko yacu twifashishije PCR yongerewe imbaraga za ziriya A. atra zikurikiranye (Ishusho 3).Mu buryo nk'ubwo, ukurikije ko genoside ya mitochondrial ya A. atra iboneka mububiko rusange, umuntu ashobora kubona ibimenyetso byerekana ko hari uduce duto twa ccfDNA ya mitochondial muri hemolymph ya A. atra.Kuba hari ibice bya ADN ya mitochondial byemejwe na PCR yongerewe imbaraga (Ishusho 3).
Ingirabuzima fatizo zitandukanye za mitochondrial zari zihari muri hemolymph ya A. atra (utudomo dutukura - nimero yimigabane: SRX5705969) na M. platensis (utudomo twubururu - nimero yimigabane: SRX5705968) yongerewe na PCR.Igishushanyo cyakuwe muri Breton nabandi, 2011 B Amplification ya hemolymph ndengakamere kuva A. atra Yabitswe ku mpapuro za FTA.Koresha mm 3 punch kugirango wongere muburyo butaziguye PCR irimo kuvanga PCR.
Urebye ibintu byinshi bya mikorobe mu mazi yo mu nyanja, twabanje kwibanda ku kuranga mikorobe ikurikirana ya hemolymph.Kugirango ukore ibi, dukoresha ingamba ebyiri zitandukanye.Ingamba ya mbere yakoresheje Kraken2, porogaramu ishingiye kuri algorithm ishingiye kuri algorithm ishobora kumenya mikorobe ikurikirana neza neza ugereranije na BLAST nibindi bikoresho [28].Ibisomwa birenga 6719 byemejwe ko bikomoka kuri bagiteri, mugihe 124 na 64 byaturutse kuri archaea na virusi (Ishusho 4).Ibice byinshi bya ADN bya bagiteri ni Firmicute (46%), Proteobacteria (27%), na Bacteroidetes (17%) (Ishusho 4a).Isaranganya rihuye nubushakashatsi bwibanze bwa microbiome yo mu nyanja yubururu [39, 40].Gammaproteobacteria yari icyiciro nyamukuru cya Proteobacteria (44%), harimo na Vibrionales nyinshi (Ishusho 4b).Uburyo bwa ddPCR bwemeje ko ibice bya ADN bya Vibrio muri ccfDNA ya A. atra hemolymph (Ishusho 4c) [41].Kugirango ubone ibisobanuro birambuye kubyerekeye inkomoko ya bagiteri ya ccfDNA, hafashwe ubundi buryo (Ishusho S2, Amakuru yinyongera). Muri uru rubanza, soma ko guhuzagurika byakusanyirijwe hamwe bisomwa-bisomwe kandi byashyizwe mu rwego rwo kwikunda (bivalves) cyangwa inkomoko ubwayo ukoresheje BLASTN hamwe na e agaciro ka 1e - 3 no guca hamwe na> 90% homology. Muri uru rubanza, soma ko guhuzagurika byakusanyirijwe hamwe bisomwa-bisomwe kandi byashyizwe mu rwego rwo kwikunda (bivalves) cyangwa inkomoko ubwayo ukoresheje BLASTN hamwe na e agaciro ka 1e - 3 no guca hamwe na> 90% homology. В этом случае перекрывающиеся ч чения были собрани как чтения с парными концами и были мсифицирован как собственные (двустворч че моллюски) или чужие по происхождению с использо зе %. Muri iki kibazo, ibyasomwe byuzuye byakusanyirijwe hamwe bisomwe byombi kandi byashyizwe mubikorwa kavukire (bivalve) cyangwa bitari umwimerere ukoresheje BLASTN na e agaciro ka 1e-3 no guca hamwe na> 90% homology.在 这种 情况 下 ， 的 读数 组装 为 配对 末端 读数 ， 并 BLASTN 和 1e-3 的 e 值 和> 90% 同源在 这 种 情况 下 ， 重叠 组装 组装 配 末端 读数 ， 使用 使用 使用 使用 使用 1e-3 的 的 值 和> 90% 同源 性 的 分类 自身 （双 壳 类） 非 自身 。。。。。。。。。 В э э случае перекрающиевающаия у чобстванны Кактыенныеннычатые Мо ллюски) Несобственнненныеныениююю Muri iki kibazo, ibisomwa byuzuye byakusanyirijwe hamwe nkibisomwa-bisomwe kandi bishyirwa mubyabo (bivalves) cyangwa bitari umwimerere ukoresheje e BLASTN na 1e-3 indangagaciro hamwe na homology imbibi> 90%.Kubera ko genoside ya A. atra itarakurikiranwa, twakoresheje ingamba zo guteranya de novo ya MEGAHIT Next Generation Sequencing (NGS).Igiteranyo cya 147.188 byagaragaye ko biterwa (bivalves) inkomoko.Aya marushanwa yahise aturika hamwe na e-gaciro ya 1e-10 ukoresheje BLASTN na BLASTX.Izi ngamba zadushoboje kumenya ibice 482 bitari bivalve biboneka muri A. atra ccfDNA.Kurenga kimwe cya kabiri (57%) by'ibi bice bya ADN byakuwe muri bagiteri, cyane cyane muri gill symbionts, harimo na sulfotrophique, ndetse no muri gill symbionts Solemya velum (Ishusho 5).
Ubwinshi bugereranije kurwego rwubwoko.B Microbial itandukanye ya phyla ebyiri nyamukuru (Firmicute na Proteobacteria).Guhagararirwa guhagararira ddPCR C Vibrio spp.A. Ibice bya 16S rRNA gene (ubururu) muri atra hemolymphs eshatu.
Hasesenguwe abantu 482 bakusanyije.Umwirondoro rusange wo gukwirakwiza tagisi ya metagenomic contig annotations (prokaryote na eukaryote).B Gukwirakwiza birambuye ibice bya ADN ya bagiteri byagaragajwe na BLASTN na BLASTX.
Isesengura rya Kraken2 ryerekanye kandi ko mussel ccfDNA yarimo ibice bya ADN bya kera, harimo ibice bya ADN bya Euryarchaeota (65%), Crenarchaeota (24%), na Thaurmarcheota (11%) (Ishusho 6a).Kuba hari ibice bya ADN byakomotse kuri Euryarchaeota na Crenarchaeota, byahoze biboneka mu muryango wa mikorobe ya mitsi ya Californiya, ntibikwiye gutungurwa [42].Nubwo Euryarchaeota ikunze kuba ifitanye isano nikirere gikabije, ubu bimaze kumenyekana ko Euryarchaeota na Crenarcheota byombi biri muri prokaryoti ikunze kugaragara mu bidukikije byo mu nyanja [43, 44].Kuba mikorobe ya methanogeneque iba muri mussele ntabwo bitangaje, urebye amakuru aherutse gutangazwa avuga ko metani nyinshi yamenetse iva mu kibaya cya Kerguelen [45] ndetse n’umusaruro wa metani wa mikorobe ushobora kugaragara ku nkombe z’ibirwa bya Kerguelen [46].
Ibitekerezo byacu byahise byerekeza kubisomwa na virusi ya ADN.Dukurikije ubumenyi dufite, ubu nubushakashatsi bwambere butagamije kumenya virusi ya mussele.Nkuko byari byitezwe, twasanze ibice bya ADN bya bacteriofage (Caudovirales) (Ishusho 6b).Nyamara, ADN ikunze kwibasira virusi ikomoka kuri phylum ya nucleocytovirus, izwi kandi nka virusi ya cytoplasme nini nini ya ADN (NCLDV), ifite genome nini ya virusi iyo ari yo yose.Muri iyi phylum, ibyiciro byinshi bya ADN ni iby'imiryango Mimimidoviridae (58%) na Poxviridae (21%), abafite ibinyabuzima bisanzwe birimo intanga ngabo na arthropodes, mu gihe igice gito cy'uruhererekane rwa ADN ari icy'imisemburo izwi ya virusi.Yanduza algae marine eukaryotic.Urukurikirane rwabonetse kandi kuri virusi ya Pandora, virusi nini ifite ubunini bunini bwa genera izwi.Igishimishije, urutonde rwabakiriye bizwi ko banduye virusi, nkuko byagenwe na hemolymph ccfDNA ikurikirana, yari nini cyane (Igicapo S3, Amakuru yinyongera).Harimo virusi zanduza udukoko nka Baculoviridae na Iridoviridae, ndetse na virusi zanduza amibe, algae na vertebrates.Twabonye kandi urutonde ruhuye na genoside ya Pithovirus sibericum.Pitovirusi (izwi kandi nka “virusi ya zombie”) yatandukanijwe bwa mbere na permafrost imaze imyaka 30.000 muri Siberiya [47].Rero, ibisubizo byacu bihuye na raporo zabanjirije iyi zerekana ko amoko yose agezweho ya virusi atazimye [48] kandi ko izo virusi zishobora kuba ziri mu bidukikije byo mu nyanja ya subarctic.
Hanyuma, twagerageje kureba niba dushobora kubona ibice bya ADN biva mu yandi matungo menshi.Abagera kuri 482 b’abanyamahanga bamenyekanye na BLASTN na BLASTX bafite amasomero ya nt, nr na RefSeq (genomic na protein).Ibisubizo byacu byerekana ko mubice byamahanga bya ccfDNA yinyamanswa zinyuranye ADN yamagufwa yamagufa yiganje (Ishusho 5).Ibice bya ADN biva mu dukoko no mu yandi moko nabyo byabonetse.Igice kinini ugereranije nuduce twa ADN ntikiramenyekana, bishoboka ko biterwa no kuba umubare munini w’ibinyabuzima byo mu nyanja biri mu bubiko bwa genomic ugereranije n’ibinyabuzima byo ku isi [49].
Muri iyi nyandiko, dushyira mu bikorwa igitekerezo cya LB kuri mussel, tuvuga ko urukurikirane rw'amasasu ya hemolymph ccfDNA rushobora gutanga ubushishozi ku bigize urusobe rw'ibinyabuzima byo ku nyanja.By'umwihariko, twasanze 1) mussel hemolymph irimo intungamubiri nyinshi (urugero rwa microgramme) zingana nini (~ 1-5 kb) zizenguruka ibice bya ADN;2) ibyo bice bya ADN byigenga kandi ntibigenga 3) Mu masoko y’amahanga y’ibi bice bya ADN, twasanze ADN ya bagiteri, archaeal na virusi, ndetse na ADN y’izindi nyamaswa nyinshi;4) Iyegeranya ryibi bice bya ccfDNA mumahanga muri hemolymph bibaho byihuse kandi bigira uruhare mubikorwa byo gushungura imbere mumitsi.Mu gusoza, ubushakashatsi bwacu bwerekana ko igitekerezo cya LB, kugeza ubu kimaze gukoreshwa cyane cyane mubijyanye na biomedicine, gikubiyemo isoko yubumenyi ikungahaye ariko idashakishwa ishobora gukoreshwa mugusobanukirwa neza imikoranire yubwoko bwa sentinel nibidukikije.
Usibye primates, hagaragaye ubwigunge bwa ccfDNA mu nyamaswa z’inyamabere, harimo imbeba, imbwa, injangwe n'amafarasi [50, 51, 52].Ariko, kubumenyi bwacu, ubushakashatsi bwacu nubwa mbere bwo kumenyekanisha gutahura no gukurikiranwa kwa ccfDNA mu moko yo mu nyanja hamwe na sisitemu yo kuzenguruka.Iyi miterere idasanzwe hamwe nubushobozi bwo kuyungurura imitsi irashobora, byibura igice, gusobanura ubunini butandukanye buranga ibice bya ADN ugereranije nandi moko.Mu bantu, ibice byinshi bya ADN bizenguruka mu maraso ni uduce duto dufite ubunini kuva kuri 150 kugeza 200 bp.hamwe nimpinga ntarengwa ya 167 bp [34, 53].Igice gito ariko gikomeye cyibice bya ADN kiri hagati ya 300 na 500 bp mubunini, naho hafi 5% ni birenga 900 bp.[54].Impamvu yo gukwirakwiza ingano ni uko isoko nyamukuru ya ccfDNA muri plasma ibaho biturutse ku rupfu rw'utugingo ngengabuzima, haba ku rupfu rw'uturemangingo cyangwa bitewe na necrosis yo kuzenguruka ingirabuzimafatizo ya hematopoietic ku bantu bafite ubuzima bwiza cyangwa bitewe na apoptose y'uturemangingo tw’ibibyimba ku barwayi ba kanseri (bizwi nka ADN ikwirakwiza ibibyimba ADN)., ctDNA).Ingano yo gukwirakwiza hemolymph ccfDNA twasanze muri mussele yari hagati ya 1000 na 5000 bp, byerekana ko ccfDNA ya mussel ifite inkomoko itandukanye.Iyi ni hypothesis yumvikana, kubera ko imitsi ifite sisitemu yimitsi ifunguye kandi ikaba mubidukikije byo mu mazi yo mu nyanja irimo ibintu byinshi bya ADN ya mikorobe.Mubyukuri, ubushakashatsi bwakozwe muri laboratoire dukoresheje ADN idasanzwe bwerekanye ko imitsi ikusanya ibice bya ADN mumazi yinyanja, byibuze nyuma yamasaha make byangiritse nyuma yo gufata selile na / cyangwa kurekurwa no / cyangwa kubikwa mumiryango itandukanye.Urebye gake ingirabuzimafatizo (zaba prokaryyotike na eukaryotic), gukoresha ibice byinjira mu nda bizagabanya umubare wa ccfDNA uva ku nkomoko ndetse no mu mahanga.Twihweje akamaro k'ubudahangarwa bw'umubiri bivalve n'umubare munini wa fagocytes ikwirakwira, twakomeje gushidikanya ko na ccfDNA yo mu mahanga ikungahaye cyane mu kuzenguruka fagocytes ikusanya ADN yo mu mahanga imaze kwinjiza mikorobe na / cyangwa imyanda ya selile.Dufatiye hamwe, ibisubizo byacu byerekana ko bivalve hemolymph ccfDNA ari ububiko bwihariye bwamakuru ya molekile kandi bishimangira umwanya wabo nkubwoko bwa sentinel.
Amakuru yacu yerekana ko gukurikirana no gusesengura ibice bikomoka kuri bagiteri ikomoka kuri hemolymph ccfDNA ishobora gutanga amakuru yingenzi kubyerekeranye na flora ya bagiteri yakira na bagiteri ziboneka mubidukikije bikikije inyanja.Uburyo bukurikirana bwo kurasa bwerekanye urutonde rwa bagiteri zisanzwe A. atra gill yaba yarabuze iyo uburyo busanzwe bwo kumenyekanisha 16S rRNA bwakoreshejwe, kuberako kubogama kubitabo bibogamye.Mubyukuri, gukoresha amakuru ya LB yakusanyirijwe muri M. platensis murwego rumwe rwimitsi i Kerguelen yerekanaga ko ibigize za bagiteri ziterwa na gill zifitanye isano na gill byari bimwe kumoko yombi yimitsi (Igicapo S4, Amakuru yinyongera).Uku guhuza imitsi ibiri itandukanye ya genetike irashobora kwerekana imiterere yabaturage ba bagiteri mububiko bukonje, sulfure, nibirunga bya Kerguelen [55, 56, 57, 58].Urwego rwo hejuru rwa mikorobe igabanya sulfuru rwasobanuwe neza mugihe cyo gusarura imitsi iva ku nkombe z’ibinyabuzima [59], nko ku nkombe za Port-au-France.Ikindi gishoboka nuko flora ya mussel flora ishobora kwanduzwa no kwandura gutambitse [60, 61].Harakenewe ubushakashatsi bwinshi kugirango hamenyekane isano iri hagati y’ibidukikije byo mu nyanja, hejuru y’inyanja, hamwe na bagiteri ya symbiotic bacteri muri mussele.Ubu bushakashatsi burakomeje.
Uburebure hamwe nubushuhe bwa hemolymph ccfDNA, ubworoherane bwo kwezwa, hamwe nubwiza buhebuje kugirango amasasu akurikirane byihuse ni bimwe mubyiza byinshi byo gukoresha mussel ccfDNA kugirango dusuzume ibinyabuzima bitandukanye mubinyabuzima byo ku nyanja.Ubu buryo ni ingirakamaro cyane cyane mu kuranga virusi (virusi) muri ecosystem runaka [62, 63].Bitandukanye na bagiteri, archaea, na eukaryote, genome za virusi ntizifite ingirabuzimafatizo zabitswe nka 16S zikurikirana.Ibisubizo byacu byerekana ko ibinyabuzima byamazi biva mu bwoko bwerekana nka mussele bishobora gukoreshwa kugira ngo hamenyekane umubare munini w’ibice bya virusi ya ccfDNA izwiho kwanduza abayituye ubusanzwe batuye urusobe rw’ibinyabuzima byo ku nyanja.Ibi birimo virusi zizwiho kwanduza protozoa, arthropodes, udukoko, ibimera, na virusi ya bagiteri (urugero, bagiteri).Isaranganya risa naryo ryabonetse mugihe twasuzumaga virusi ya hemolymph ccfDNA ya musele yubururu (M. platensis) yakusanyirijwe murwego rumwe rwimitsi i Kerguelen (Imbonerahamwe S2, Amakuru yinyongera).Urukurikirane rw'amasasu ya ccfDNA mubyukuri nuburyo bushya bugenda bwiyongera mubushakashatsi bwa virusi yabantu cyangwa ubundi bwoko [21, 37, 64].Ubu buryo ni ingirakamaro cyane cyane mu kwiga virusi ya ADN ifite imirongo ibiri, kubera ko nta gene imwe ibikwa muri virusi zose za ADN ebyiri, zerekana ubwoko bwa virusi butandukanye kandi bwagutse muri Baltimore [65].Nubwo inyinshi muri izo virusi ziguma zidafite urutonde kandi zishobora kuba zirimo virusi ziva mu gice kitazwi na gato ku isi ya virusi [66], twasanze virusi hamwe n’urwego rwakira imitsi A. atra na M. platensis bigwa hagati yubwoko bubiri.kimwe (reba ishusho S3, amakuru yinyongera).Ibi bisa ntabwo bitangaje, kuko bishobora kwerekana kubura guhitamo gufata ADN iboneka mubidukikije.Inyigisho z'ejo hazaza ukoresheje RNA isukuye irakenewe kuri ubu kuranga virusi ya RNA.
Mu bushakashatsi bwacu, twakoresheje umuyoboro ukomeye cyane wakuwe mu mirimo ya Kowarski na bagenzi be [37], bakoresheje gusiba intambwe ebyiri zo gusoma hamwe hamwe na kontig mbere na nyuma yo guteranya ccfDNA kavukire, bigatuma umubare munini w'abasoma badashushanyije.Ntabwo rero, ntidushobora kwirengagiza ko bimwe muribi bisomwa bidasomwe bishobora kuba bigifite inkomoko yabyo, cyane cyane ko tudafite genome yerekeranye nubwoko bwimitsi.Twakoresheje kandi uyu muyoboro kuko twahangayikishijwe na chimeras hagati yo gusoma no kutigenga ndetse n'uburebure bwo gusoma bwakozwe na Illumina MiSeq PE75.Indi mpamvu yatumye abantu benshi basoma badasobanuwe ni uko mikorobe nyinshi zo mu nyanja, cyane cyane mu turere twa kure nka Kerguelen, zitigeze zitangazwa.Twakoresheje Illumina MiSeq PE75, tuvuze ko uburebure bwa ccfDNA busa na ccfDNA yabantu.Kubushakashatsi buzaza, ukurikije ibisubizo byacu byerekana ko hemolymph ccfDNA imaze gusoma cyane kurusha abantu na / cyangwa inyamaswa z’inyamabere, turasaba ko dukoresha urubuga rukurikirana rukwiranye n’ibice birebire bya ccfDNA.Iyi myitozo izoroha cyane kumenya ibimenyetso byinshi byo gusesengura byimbitse.Kubona ibyuzuye A. atra byuzuye bya genome bikurikirana nabyo byoroshya cyane ivangura rya ccfDNA bivuye kubiturutseho kandi bitari ubwabo.Bitewe nuko ubushakashatsi bwacu bwibanze ku buryo bwo gushyira mu bikorwa igitekerezo cya biopsy y’amazi kuri mussele, turizera ko nkuko iki gitekerezo kizakoreshwa mu bushakashatsi buzaza, hazashyirwaho ibikoresho bishya n’imiyoboro kugira ngo byongere ubushobozi bw’ubu buryo bwo kwiga mikorobe zitandukanye z’imitsi.urusobe rw'ibinyabuzima byo mu nyanja.
Nka biomarker yubuvuzi idatera, hejuru ya plasma yabantu ya ccfDNA ifitanye isano nindwara zitandukanye, kwangirika kwinyama, hamwe nuburwayi [67,68,69].Uku kwiyongera gufitanye isano no kurekura ibice bya ADN inkomoko yabyo nyuma yo kwangirika kwinyama.Twakemuye iki kibazo dukoresheje ubushyuhe bukabije, aho imitsi yahuye nubushyuhe bwa 30 ° C.Twakoze isesengura ryubwoko butatu bwa mussele mubushakashatsi butatu bwigenga.Ariko, ntabwo twabonye impinduka murwego rwa ccfDNA nyuma yubushyuhe bukabije (reba Ishusho S5, amakuru yinyongera).Ubu buvumbuzi bushobora gusobanura, byibuze igice, kuba imitsi ifite sisitemu yo gutembera igice cya kabiri kandi ikusanya ADN nyinshi zamahanga kubera ibikorwa byazo byo kuyungurura.Ku rundi ruhande, imitsi, kimwe n’inyamaswa nyinshi zidafite ubuzima, zishobora kwihanganira kwangirika kwangirika kw ingirabuzimafatizo, bityo bikagabanya irekurwa rya ccfDNA muri hemolymph yabo [70, 71].
Kugeza ubu, isesengura rya ADN ry’ibinyabuzima mu binyabuzima byo mu mazi byibanze cyane cyane kuri ADN y’ibidukikije (eDNA) metabarcoding.Nyamara, ubu buryo busanzwe bugarukira mubisesengura ryibinyabuzima iyo primers ikoreshwa.Gukoresha imbunda zikurikirana bizenguruka imipaka ya PCR no guhitamo kubogamye kwa primer set.Rero, muburyo bumwe, uburyo bwacu buregereye uburyo bukoreshwa bwa vuba bwa eDNA Shotgun uburyo bukurikirana, bushobora gukurikirana ADN yacitsemo ibice no gusesengura ibinyabuzima hafi ya byose [72, 73].Ariko, hariho ibibazo byinshi byingenzi bitandukanya LB nuburyo busanzwe bwa eDNA.Birumvikana, itandukaniro nyamukuru hagati ya eDNA na LB nugukoresha ibiyungurura bisanzwe.Ikoreshwa ry’amoko yo mu nyanja nka sponges na bivalves (Dresseina spp.) Nkayunguruzo karemano yo kwiga eDNA byavuzwe [74, 75].Nyamara, ubushakashatsi bwa Dreissena bwakoresheje tissue biopsies yakuwemo ADN.Isesengura rya ccfDNA riva muri LB ntirisaba biopsy ya tissue, ibikoresho byihariye kandi rimwe na rimwe bihenze hamwe nibikoresho bifitanye isano na eDNA cyangwa biopsy ya tissue.Mubyukuri, duherutse gutangaza ko ccfDNA yo muri LB ishobora kubikwa no gusesengurwa ku nkunga ya FTA idakomeje urunigi rukonje, kikaba ari ikibazo gikomeye ku bushakashatsi mu turere twa kure [76].Gukuramo ccfDNA muri biopsies yamazi nabyo biroroshye kandi bitanga ADN nziza yo murwego rwo gukurikiranya imbunda no gusesengura PCR.Iyi ninyungu nini urebye zimwe mu mbogamizi tekinike zijyanye no gusesengura eDNA [77].Ubworoherane nigiciro gito cyuburyo bwo gutoranya nabyo birakwiriye cyane cyane gahunda yo gukurikirana igihe kirekire.Usibye ubushobozi bwabo bwo kuyungurura, ikindi kintu kizwi cyane kiranga bivale ni imiti ya mucopolysaccharide igizwe na mucus yabo, itera kwinjiza virusi [78, 79].Ibi bituma habaho guhuza akayunguruzo keza ko kuranga urusobe rw'ibinyabuzima n'ingaruka z'imihindagurikire y’ikirere mu bidukikije by’amazi.Nubwo kuba hari ibice bya ADN byakomotse kuri ADN bishobora kugaragara nkimbogamizi yuburyo ugereranije na eDNA, ikiguzi kijyanye no kugira ccfDNA kavukire ugereranije na eDNA birumvikana icyarimwe kubwamakuru menshi aboneka kubushakashatsi bwubuzima.kubakira.Ibi birimo kuba hari virusi zikurikirana zinjiye muri genome ya host host.Ibi ni ingenzi cyane cyane kuri mussele, urebye hariho retrovirus zandurira mu buryo butambitse muri bivalves [80, 81].Iyindi nyungu ya LB kurenza eDNA nuko ikoresha ibikorwa bya fagocyitike yo kuzenguruka ingirabuzimafatizo zamaraso muri hemolymph, ifata mikorobe (na genome).Phagocytose nigikorwa nyamukuru cyingirangingo zamaraso muri bivalves [82].Hanyuma, uburyo bwifashisha ubushobozi bwo kuyungurura hejuru ya mussele (ugereranije 1.5 l / h y'amazi yo mu nyanja) hamwe no kuzenguruka iminsi ibiri, ibyo bikaba byongera kuvanga ibice bitandukanye byamazi yinyanja, bigatuma ifatwa rya eDNA itandukanye.[83, 84].Rero, mussel ccfDNA isesengura ninzira ishimishije ukurikije imirire, ubukungu, nibidukikije bya mussele.Kimwe nisesengura rya LB ryakusanyirijwe mu bantu, ubu buryo kandi burafungura amahirwe yo gupima ihinduka ry’imiterere n’irondakoko muri ADN yakiriye hasubijwe ibintu bidasanzwe.Kurugero, tekinoroji yo mu gisekuru cya gatatu irashobora gutegurwa gukora genome-methylation isesengura muri ccfDNA kavukire ikoresheje nanopore ikurikirana.Iyi nzira igomba koroherezwa nuko uburebure bwibice bya ccfDNA ya mussel bihuza neza nibisomwa byasomwe igihe kirekire byemerera isesengura rya methylation ya ADN ya genome yagutse uhereye kumurongo umwe ukenera bidakenewe ko hahindurwa imiti.Niyo mpamvu, irashobora gutanga ubumenyi bwingenzi muburyo bwihuse bwo kugenzura igisubizo nyuma y’imihindagurikire y’ikirere cyangwa umwanda [87].Ariko, gukoresha LB ntabwo bigarukira.Ntawabura kuvuga, ibi bisaba ko habaho ubwoko bwibipimo byerekana ibidukikije.Nkuko byavuzwe haruguru, gukoresha LB kugirango usuzume urusobe rwibinyabuzima by’ibinyabuzima runaka bisaba kandi umuyoboro ukomeye wa bioinformatics uzirikana ko hari ibice bya ADN biva aho biva.Ikindi kibazo gikomeye nukuboneka genome zikoreshwa kubinyabuzima byo mu nyanja.Twizera ko ibikorwa nk'umushinga wa Marine Mammal Genomes n'umushinga Fish10k uherutse gushingwa [88] bizorohereza isesengura nk'iryo mu bihe biri imbere.Gushyira mu bikorwa igitekerezo cya LB ku binyabuzima bigaburira mu nyanja na byo birahuza n’iterambere rigezweho mu ikoranabuhanga rikurikirana, bigatuma bikwiranye n’iterambere ry’ibinyabuzima byinshi-ohm kugira ngo bitange amakuru yingenzi ku buzima bw’imiterere y’inyanja hagamijwe guhangana n’ibidukikije.
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