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1.人獸共患寄生蟲

1.1A Fluorescent Recombinase Aided AmplificationAssay for Detection of Babesia microt

1.2Rapid Visual Detection of Plasmodium Using Recombinase-Aided Amplification With Lateral Flow DipstickAssay

1.3重組酶介導(dǎo)恒溫?cái)U(kuò)增技術(shù)檢測瘧原蟲方法的建立

1.4實(shí)時(shí)熒光重組酶介導(dǎo)核酸擴(kuò)增在瘧原蟲快速檢測中的應(yīng)用研究

1.5重組酶介導(dǎo)的斯氏并殖吸蟲等溫?cái)U(kuò)增熒光檢測方法的建立及檢測效果初步評(píng)價(jià)

1.6重組酶介導(dǎo)的日本血吸蟲特異性基因片段核酸等溫?cái)U(kuò)增檢測方法的建立

1.7結(jié)合重組酶介導(dǎo)的核酸等溫?cái)U(kuò)增和熒光探針快速檢測日本血吸蟲基因片段

1.8加強(qiáng)分子診斷方法的研發(fā)應(yīng)用助力我國精準(zhǔn)血防

1.9重組酶介導(dǎo)的核酸等溫?cái)U(kuò)增熒光法快速檢測日本血吸蟲感染性釘螺

1.10重組酶介導(dǎo)的隱孢子蟲屬特異性等溫核酸擴(kuò)增方法的建立及評(píng)價(jià)

1.11重組酶介導(dǎo)的華支睪吸蟲特異性核酸等溫?cái)U(kuò)增方法的建立及初步評(píng)價(jià)

1.12再送“瘟神”:消除血吸蟲病適宜技術(shù)最新研究進(jìn)展

1.13重組酶介導(dǎo)的等溫核酸擴(kuò)增技術(shù)檢測多房棘球絳蟲方法的建立及初步應(yīng)用

1.14重組酶介導(dǎo)等溫核酸擴(kuò)增技術(shù)檢測細(xì)棘球絳蟲方法的建立及初步應(yīng)用評(píng)價(jià)

1.15基于重組酶介導(dǎo)等溫?cái)U(kuò)增技術(shù)的廣州管圓線蟲核酸檢測方法的建立

1.16基于重組酶介導(dǎo)等溫?cái)U(kuò)增技術(shù)的細(xì)粒棘球絳蟲核酸檢測方法的建立

1.17基于重組酶介導(dǎo)核酸等溫?cái)U(kuò)增反應(yīng)的曼氏血吸蟲基因檢測方法的建立

1.18新型等溫?cái)U(kuò)增技術(shù)推動(dòng)寄生蟲病現(xiàn)場快速檢測能力提升

1.19重組酶介導(dǎo)的藍(lán)氏賈第鞭毛蟲特異性等溫核酸擴(kuò)增方法的建立及評(píng)價(jià)

1.20基于熒光重組酶介導(dǎo)等溫?cái)U(kuò)增技術(shù)的利什曼原蟲核酸檢測方法的建立

1.21基于重組酶介導(dǎo)核酸等溫?cái)U(kuò)增技術(shù)的日本血吸蟲特異性基因片段核酸試紙條檢測方法的建立

1.22快速檢測田鼠巴貝西蟲重組酶介導(dǎo)核酸等溫?cái)U(kuò)增方法的建立

1.23 重組酶介導(dǎo)的核酸等溫?cái)U(kuò)增熒光法檢測日本血吸蟲感染性釘螺的效能評(píng)價(jià)

1.24重組酶介導(dǎo)等溫?cái)U(kuò)增技術(shù)檢測瘧原蟲方法的建立和評(píng)價(jià)

1.25重組酶介導(dǎo)核酸等溫?cái)U(kuò)增熒光法用于日本血吸蟲感染性釘螺旱期檢測的研究

A Fluorescent Recombinase Aided Amplifica-tion Assay for Detection of Babesia microti

Abstract Babesia microti is one of the most common causative agents of babesiosis.A sensitive and raplddetection is necessary for screening potentially infected individuals.In this study,B. microti cytochrome coxldase subunit l(cox1) was selected as the target gene,multiple primers were designed,and optimized by arecombinase-aided amplification(RAA) assay.The optimal primers and probe were labeled with fluorescein.The sensitivity of fluorescent RAAGRAA) was evaluated using gradient diluents of the cox1 recombinantplasmid and genomic DNA extracted from whole blood of B.microti infected mice.The spedificity offRAA wasassessed by other transfusion transmitted parasites.The analytical sensitivity of the fRAA assay was 10 coplesof recombinant plasmid per reaction and 10 fg/ul B.microti genomic DNA.No cross-re action with any otherblood-transmitted parasites was observed.Our results demonstrated that the fRAA assay would be rapid,sensitive,and specific for the detection of B.microti.

Key words:Babesia microti,recombinase-aided amplification,molecular detection.

Rapid Visual Detection ofPlasmodium Using Recombinase Aided Am-plification With Lateral Flow Dipstick Assay

Background:Malaria is a global public health problem.China has had no case of indigenous malaria since 2016.Hawever,imported cases of malaria remain an issue amang travelers,overseas workers,and foreign traders.Although these cases are always asymptomatic,if they donate blood,there is a great risk of transfusion trans-mitted-malaria(TTM).Therefore,blood banks need a rapid screening tool to detect Pasmodium species.Methods:We designed an assay using recombinase-aided amplification(RAA) and a lateral-flow dipstick(LFD)(RAA-LFD) to detect the 18S ribasomal RNA gene of Plasmodium species.Sensitivity was evaluated using arecombinant plasmid and Plasmodium genomic DNA.Specificity was evaluated using DNA extracted from theblood of patients with malaria ar other infectious parasites.For clinical assessment,blood samples frompatients with malaria and blood donors were evalu ated.Results:The RAA-LFD assay was performed in an incubator block at 37C for 15 min,and the amplicans werevisible to the naked eye an the flaw dipsticks within 3 min.The sensitivity was 1 copy/mL of recombinantplasmid.For genomic DNA from whole blood of malaria patients infected with P.falciparum, P.vivax,P. ovale,and P.malariae,the sensitivity was0.1 pg/mL,10 pg/mL,10-100 pg/mL,and 100pg/mL,respectively.Thesensi-tivity of this assay was 100pg/mL.No cross-reaction withother transfusion transmissible parasites wasdetect-ed.Conclusions:The results demonstrated that this RAA-LFD assay was suitable for reliable field detection ofPlasmodium spedes in low-resource settings with limited laboratory capabilities.

Keywords:malaria,nucleic add detection,plasmodium,recombinase-aided amplification, lateralflow dipstick.

重組酶介導(dǎo)恒溫?cái)U(kuò)增技術(shù)檢測瘧原蟲方法的建立

摘要：本研究利用重組酶介導(dǎo)恒溫?cái)U(kuò)增RAA技術(shù)，選取瘧原蟲18SrDNA序列，設(shè)計(jì)了檢測瘧原蟲的通用引物，并對(duì)引物進(jìn)行篩選和對(duì)其特異性進(jìn)行檢測。篩選出4組擴(kuò)增效果較好的引物。該方法整個(gè)反應(yīng)過程在37℃下進(jìn)行，擴(kuò)增時(shí)間短(40min)，并具有良好的特異性。成功建立了一種檢測瘧原蟲的RAA法，并且該方法適合于進(jìn)出口岸瘧原蟲的快速檢測。

關(guān)鍵詞：重組酶介導(dǎo)擴(kuò)增；瘧原蟲；分子檢測；瘧疾。

重組酶介導(dǎo)的斯氏并殖吸蟲等溫?cái)U(kuò)增熒光檢測方法的建立及檢測效果初步評(píng)價(jià)

摘要：目的建立一種基于重組酶介導(dǎo)的等溫?cái)U(kuò)增(recombinase-aided isothermal amplific ation,RAA)技術(shù)的斯氏并殖吸蟲快速核酸檢測方法，并對(duì)其檢測效果進(jìn)行初步評(píng)價(jià)。方法從溪蟹樣本中分離出斯氏并殖吸蟲、衛(wèi)氏并殖吸蟲和三平正并殖吸蟲囊坳,提取基因組DNA進(jìn)行分子鑒定。以斯氏并殖吸蟲線粒體細(xì)胞色素c氧化酶亞基I基因(cytochrome coxidase 1, cox1)基因序列作為靶序列設(shè)計(jì)、制備、篩選引物及探針。以河南省濟(jì)源市和洛陽市宜陽縣斯氏并殖吸蟲囊蛻基因組DNA為模板進(jìn)行熒光RAA檢測方法驗(yàn)證。以含斯氏并殖吸蟲cox1基因序列的不同濃度重組質(zhì)粒和斯氏并殖吸蟲囊坳基因組DNA為模板進(jìn)行熒光RAA擴(kuò)增，評(píng)價(jià)其檢測靈敏度；應(yīng)用建立的熒光RAA法同時(shí)檢測衛(wèi)氏并殖吸蟲、三平正并殖吸蟲、華支睪吸蟲和日本血吸蟲基因組DNA，評(píng)價(jià)其檢測特異性。結(jié)果從溪蟹樣本中分離出斯氏并殖吸蟲、衛(wèi)氏并殖吸蟲和三平正并殖吸蟲囊蝴，經(jīng)分子鑒定和系統(tǒng)進(jìn)化分析確認(rèn)其與GenBank中并殖吸蟲標(biāo)準(zhǔn)株基因序列具有同源性。成功建立了斯氏并殖吸蟲熒光RAA檢測方法，可在5min內(nèi)擴(kuò)增到河南省濟(jì)源市和洛陽市宜陽縣采集的斯氏并殖吸蟲囊鮒基因組DNA，而陰性對(duì)照無擴(kuò)增。以重組質(zhì)粒為模板,熒光RAA法最低檢出限為10拷貝/L重組質(zhì)粒,且均在5 min內(nèi)出現(xiàn)陽性擴(kuò)增；以基因組DNA為模板,可檢測到的最低模板DNA濃度為10 pgluL，且均在10~15min內(nèi)出現(xiàn)陽性擴(kuò)增。熒光RAA法檢測衛(wèi)氏并殖吸蟲、三平正并殖吸蟲、日本血吸蟲和華支睪吸蟲基因組DNA結(jié)果均為陰性。結(jié)論成功建立了一種基于熒光RAA技術(shù)的快速、敏感、特異的斯氏并殖吸蟲核酸檢測方法,在斯氏并殖吸蟲病流行區(qū)溪蟹現(xiàn)場快速檢測與蟲種鑒定中具有潛在應(yīng)用價(jià)值。

關(guān)鍵詞：斯氏并殖吸蟲；重組酶介導(dǎo)的等溫?cái)U(kuò)增；核酸檢測；檢測效果。