本文關(guān)鍵詞：云南省惡性瘧原蟲(chóng)氯喹及青蒿素抗性相關(guān)基因的聯(lián)合突變分析　出處：《中國(guó)寄生蟲(chóng)學(xué)與寄生蟲(chóng)病雜志》2017年03期 　論文類(lèi)型：期刊論文

  更多相關(guān)文章： 云南省 惡性原瘧蟲(chóng) 氯喹抗性 青蒿素抗性 氯喹轉(zhuǎn)運(yùn)蛋白基因 青蒿素抗性相關(guān)基因 聯(lián)合突變 分析

【摘要】：目的分析云南省惡性瘧原蟲(chóng)抗氯喹轉(zhuǎn)運(yùn)蛋白(Plasmodium falciparum chloroquine resistant transporter,Pfcrt)基因和青蒿素抗性相關(guān)(K13)基因的聯(lián)合突變特性。方法收集2012年8月-2015年12月寄生蟲(chóng)病防治信息管理系統(tǒng)登記和報(bào)告的云南省惡性瘧病例濾紙血樣和流行病學(xué)史等相關(guān)信息,提取瘧原蟲(chóng)DNA,巢式PCR擴(kuò)增惡性瘧原蟲(chóng)Pfcrt基因exon2區(qū)和K13基因kelch結(jié)構(gòu)域并測(cè)序,測(cè)序序列與2655199、PF3D7_1343700參比序列進(jìn)行比對(duì),用MEGA 5.04、Arlequin 3.01軟件分析Pfcrt基因exon2區(qū)、K13基因kelch結(jié)構(gòu)域的單倍型、單倍型間期望雜合度(He)及其群體間的遺傳分化指數(shù)(Fst)等,用Network 4.6.0軟件制作單倍型中介網(wǎng)絡(luò)進(jìn)化圖,采用Epi Data 3.1軟件建立數(shù)據(jù)庫(kù),SPSS 21.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)分析。結(jié)果共收集惡性瘧病例血樣234份,Pfcrt基因exon2區(qū)、K13基因kelch結(jié)構(gòu)域巢式PCR擴(kuò)增產(chǎn)物同時(shí)成功測(cè)序192份。其中,云南省本地感染病例血樣12份,自非洲、緬甸感染的病例血樣分別為30和150份。218份血樣的Pfcrt基因exon2區(qū)DNA序列有7種單倍型,He為0.238 5,錯(cuò)義突變序列占83.0%(181/218),72~76位點(diǎn)呈單重突變(CVMNT)、雙重突變(SVMNT)、三重突變(CVIET)的比例分別為1.8%(4/218)、6.4%(14/218)和74.8%(163/218),7種單倍型以氯喹敏感型CVMNK為起始,再按單重、雙重及三重突變的路徑逐步進(jìn)化。192份血樣的K13基因kelch結(jié)構(gòu)域DNA序列有21種單倍型,He為0.044 9,錯(cuò)義突變序列占35.9%(69/192),在16、446、676等9個(gè)位點(diǎn)均只發(fā)生單重突變,F446I的突變率最高,為25.0%(48/192),21種單倍型的中介網(wǎng)絡(luò)圖顯示"星狀"布局。云南省本地惡性瘧原蟲(chóng)種群與緬甸種群間的Fst最小,為0.020 3。Pfcrt基因的氯喹敏感型CVMNK血樣中,K13基因kelch結(jié)構(gòu)域位點(diǎn)突變的比例為20.6%(7/34),氯喹抗性型CVMNT、CVIET血樣中的檢出比例分別為1/4和36.4%(51/140)。結(jié)論云南省疫情報(bào)告病例中惡性瘧原蟲(chóng)的氯喹、青蒿素相關(guān)抗性基因的聯(lián)合突變率為27.1%,且青蒿素抗性基因突變型之間可能存在種群擴(kuò)張模式。
[Abstract]:Analysis of Plasmodium falciparum in Yunnan province chloroquine resistance transporter (Plasmodium falciparum chloroquine resistant to transporter Pfcrt) and artemisinin resistance related gene (K13) mutation characteristic gene. Methods from August 2012 December -2015 parasitic disease information management system for registration and report a case of falciparum malaria in Yunnan province. Blood samples and epidemiological history and other related information extraction. Plasmodium DNA, nested PCR amplification of Pfcrt gene of Plasmodium falciparum Exon2 gene region and K13 domain of kelch and sequencing, sequencing and sequence 2655199 PF3D7_1343700 reference sequence were compared with MEGA 5.04, Exon2 Arlequin 3.01 Pfcrt gene analysis software, the kelch domain of K13 gene haplotype, haplotype between the expected heterozygosity (He) genetic index differentiation and between groups (Fst), making the intermediary network evolution haplotype map with Network 4.6.0 software, using Epi Da TA 3.1 software to establish database, SPSS 21 statistical software for data analysis. The results were collected blood samples of 234 cases of malignant malaria, Pfcrt Exon2 gene, K13 gene kelch domain of nested PCR amplification products and successfully sequenced 192 copies. Among them, Yunnan province local infection and 12 blood samples were collected from Africa, Burma cases were respectively. 30 and 150.218 Exon2 region of Pfcrt gene were DNA sequence had 7 haplotypes, He 0.2385 missense mutation sequences accounted for 83% (181/218), 72~76 loci showed single mutation (CVMNT), double mutant (SVMNT), three (CVIET) mutation rates were 1.8% (4/218). 6.4% (14/218) and 74.8% (163/218), 7 haplotypes with chloroquine sensitive CVMNK as starting, according to the single, K13 kelch gene DNA domain sequences and three double mutant.192 path gradually evolved blood samples of 21 haplotypes, He 0.0449 missense mutation sequence Accounted for 35.9% (69/192), in the 16446676 and 9 loci were only single mutation, the mutation rate of F446I is the highest, was 25% (48/192), intermediary network figure 21 haplotypes showed the "star shaped" layout. Yunnan province local P.falciparum populations between Burma population and the minimum is 0.020 Fst, 3.Pfcrt gene chloroquine sensitive CVMNK in blood, K13 gene kelch domain mutation ratio was 20.6% (7/34), chloroquine resistant type CVMNT detection ratio of CVIET in blood samples were 1/4 and 36.4% (51/140). Conclusion the Plasmodium falciparum epidemic reporting cases in Yunnan Province, the resistance gene of chloroquine, artemisinin related mutation rate was 27.1% there may be a population expansion mode, and between artemisinin resistance gene mutant.

【作者單位】： 云南省寄生蟲(chóng)病防治所云南省瘧疾研究中心云南省蟲(chóng)媒傳染病防控研究重點(diǎn)實(shí)驗(yàn)室;大理大學(xué)基礎(chǔ)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金國(guó)際(地區(qū))合作與交流項(xiàng)目(No.81220108019);國(guó)家自然科學(xué)基金地區(qū)科學(xué)基金項(xiàng)目(No.81660559) 云南省科技項(xiàng)目應(yīng)用基礎(chǔ)研究計(jì)劃青年項(xiàng)目(No.Y0120150295)~~
【分類(lèi)號(hào)】：R382.31
【正文快照】： kelch domain were amplified with nest-PCR.PCR products were sequenced,and the sequences were aligned with thereference sequences 2655199 and PF3D7_1343700.The haplotype number,expected heterozygosity(He),and geneticdifferentiation(fixation index,Fst)of t

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