基于线粒体细胞色素氧化酶亚单位3基因鉴定贵州省首例输入性卵形疟病例
|
摘要
提取贵州省首例镜检诊断为输入性卵形疟病例的血样DNA,以疟原虫的核糖体RNA小亚基(SSU r RNA)和线粒体细胞色素氧化酶亚单位3基因(cox3)为靶标,分别设计属、种特异性引物及种特异性引物, PCR扩增、测序,比较2种引物对疟原虫种类PCR鉴定的特异性。以卵形疟原虫wallikeri亚种参考序列(GenBank登录号:HQ712053.1)和curtisi亚种参考序列(GenBank登录号:HQ712052.1)为模板,应用DNAMAN V6软件对扩增序列进行Blast比对。利用Mega 7.0.26软件,采用邻接法,基于疟原虫线粒体cox3基因DNA序列构建系统进化树。PCR结果显示,基于疟原虫SSU r RNA属、种特异性引物扩增,仅获得1 200 bp的属特异性条带,未出现种特异性条带。基于疟原虫cox3种特异性引物扩增,可获得880 bp的目的条带。Blast比对结果显示,PCR扩增获得的cox3基因序列与卵形疟原虫wallikeri亚种和curtisi亚种的序列一致度为99.4%和97.4%。
To develop a PCR-based detection of Plasmodium ovale infection, DNA was extracted from blood of a patient diagnosed as the first imported case of P. ovale infection in Guizhou province, two sets of PCR based on the Plasmodium ribosomal RNA small subunit(SSU r RNA) and mitochondrial cytochrome oxidase subunit 3 gene(cox3) were established. The genus, species-specific primers were designed accordingly, the specificity of Plasmodium species detection was performed using the designed primers and the amplified cox3 PCR products were sequenced.The obtained sequences were aligned with reference sequence of the P. ovale wallikeri subspecies(GenBank accession number: HQ712053.1) and the curtisi subspecies( GenBank accession number: HQ712052.1) using DNAMAN V6. A phylogenetic tree was constructed based on the Plasmodium cox3 gene using the neighbor-joining method and Mega7.0.26 software. The PCR results showed that only a genus-specific band with 1 200 bp was obtained based on Plasmodium SSU r RNA and no species-specific bands were amplified. However, a PCR product with 880 bp was amplified from the patient sample based on Plasmodium cox3 gene. The Blast alignment showed that the obtained cox3 sequence shared 99.4% identity with P. ovale wallikeri subspecies and 97.4% with curtisi subspecies.
To develop a PCR-based detection of Plasmodium ovale infection, DNA was extracted from blood of a patient diagnosed as the first imported case of P. ovale infection in Guizhou province, two sets of PCR based on the Plasmodium ribosomal RNA small subunit(SSU r RNA) and mitochondrial cytochrome oxidase subunit 3 gene(cox3) were established. The genus, species-specific primers were designed accordingly, the specificity of Plasmodium species detection was performed using the designed primers and the amplified cox3 PCR products were sequenced.The obtained sequences were aligned with reference sequence of the P. ovale wallikeri subspecies(GenBank accession number: HQ712053.1) and the curtisi subspecies( GenBank accession number: HQ712052.1) using DNAMAN V6. A phylogenetic tree was constructed based on the Plasmodium cox3 gene using the neighbor-joining method and Mega7.0.26 software. The PCR results showed that only a genus-specific band with 1 200 bp was obtained based on Plasmodium SSU r RNA and no species-specific bands were amplified. However, a PCR product with 880 bp was amplified from the patient sample based on Plasmodium cox3 gene. The Blast alignment showed that the obtained cox3 sequence shared 99.4% identity with P. ovale wallikeri subspecies and 97.4% with curtisi subspecies.
引文
[1] Collins WE,Jeffery GM. Plasmodium ovale:parasite and disease[J]. Clin Microbiol Rev,2005,18(3):570-581.
[2]汤林华,许隆祺,陈颖丹.中国寄生虫病防治与研究[M].北京:北京科学技术出版社,2012:76,1345.
[3]陈玉凤,王艳艳,姜杰,等.应用巢式PCR评价低流行区疟疾诊断结果[J].国际医学寄生虫病杂志,2013,40(6):311-316.
[4]周瑞敏,张红卫,邓艳,等. 2例输入性卵形疟的实验室检测[J].中国寄生虫学与寄生虫病杂志,2013,31(2):127-130.
[5]肖婷,魏艳彬,刘凡,等.山东省2例输入性卵形疟病例报告[J].中国病原生物学杂志,2012,7(6):附页4.
[6]王晓光,雷永良,兰进权,等.浙江省1例输入性卵形疟的实验室检测分析[J].中国寄生虫学与寄生虫病杂志,2013,31(1):78-79.
[7]汤林华.输入性疟疾的诊治与管理[M].上海:上海科学技术出版社,2010:70-73.
[8]黄天谊,王世海,黎学铭,等.标签引物-套式/多重PCR检测恶性疟原虫和间日疟原虫的研究[J].中国寄生虫学与寄生虫病杂志,2005,23(3):140-142.
[9]郭传坤,黎学铭,李锦辉,等.套式/多重PCR诊断疟疾的敏感性、特异性和稳定性初探[J].中国寄生虫学与寄生虫病杂志,2007,25(3):213-216.
[10]黄雨婷,佘丹娅,卢丽丹,等.单管单轮多重PCR检测4种疟原虫混合血样[J].中国寄生虫学与寄生虫病杂志,2015,33(3):200-205.
[11] Sutherland CJ,Tanomsing N,Nolder D,et al. Two non recombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally[J]. J Infect Dis,2010,201(10):1544-1550.
[12] Haanshuus CG,Mohn SC,M?rch K,et al. A novel,singleamplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen,Norway[J]. Malar J,2013,12(1):26.
[13] Sherman IW. Malaria:parasite biology,pathogenesis,and pro tection[M] Second edition. Washington D.C:American Society for Microbiology,1998:267-270.
[14]张玲玲,阮卫,陈华良,等.浙江省5例输入性卵形疟原虫wallikeri亚种感染病例的鉴定[J].中国寄生虫学与寄生虫病杂志,2014,32(5):361-365.
[15]陈梦妮,毛祥华,邓艳,等.云南省疟疾疫情病例疟原虫镜检疑难形态的鉴定[J].中国病原生物学杂志,2017,12(3):227-232.
[16]谭妍,李志峰,凌华,等. 2015年重庆市3例输入性卵形疟原虫wallikeri亚种感染病例的实验室诊断[J].热带病与寄生虫学,2016,14(3):141-144.
[17]李美,夏志贵,汤林华.卵形疟原虫wallikeri亚种及其基因检测体系的研究进展[J].中国寄生虫学与寄生虫病杂志,2014,32(1):64-67.
[18]贾西帅,周水茂,杨燕,等.武汉市两种输入性卵形疟巢式PCR检测[J].中国热带医学,2018,18(5):423-426.
[19]张玲玲,姚立农,陈华良,等.输入性卵形疟原虫细胞色素b、细胞色素c氧化酶Ⅰ和乳酸脱氢酶基因单核苷酸多态性分析[J].中国寄生虫学与寄生虫病杂志,2017,35(5):429-433
[20]李瑾,魏庆宽,徐超,等.山东省14例输入性卵形疟原虫wallikeri亚种的鉴定[J].中国人兽共患病学报,2018,34(11):991-995.
[2]汤林华,许隆祺,陈颖丹.中国寄生虫病防治与研究[M].北京:北京科学技术出版社,2012:76,1345.
[3]陈玉凤,王艳艳,姜杰,等.应用巢式PCR评价低流行区疟疾诊断结果[J].国际医学寄生虫病杂志,2013,40(6):311-316.
[4]周瑞敏,张红卫,邓艳,等. 2例输入性卵形疟的实验室检测[J].中国寄生虫学与寄生虫病杂志,2013,31(2):127-130.
[5]肖婷,魏艳彬,刘凡,等.山东省2例输入性卵形疟病例报告[J].中国病原生物学杂志,2012,7(6):附页4.
[6]王晓光,雷永良,兰进权,等.浙江省1例输入性卵形疟的实验室检测分析[J].中国寄生虫学与寄生虫病杂志,2013,31(1):78-79.
[7]汤林华.输入性疟疾的诊治与管理[M].上海:上海科学技术出版社,2010:70-73.
[8]黄天谊,王世海,黎学铭,等.标签引物-套式/多重PCR检测恶性疟原虫和间日疟原虫的研究[J].中国寄生虫学与寄生虫病杂志,2005,23(3):140-142.
[9]郭传坤,黎学铭,李锦辉,等.套式/多重PCR诊断疟疾的敏感性、特异性和稳定性初探[J].中国寄生虫学与寄生虫病杂志,2007,25(3):213-216.
[10]黄雨婷,佘丹娅,卢丽丹,等.单管单轮多重PCR检测4种疟原虫混合血样[J].中国寄生虫学与寄生虫病杂志,2015,33(3):200-205.
[11] Sutherland CJ,Tanomsing N,Nolder D,et al. Two non recombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally[J]. J Infect Dis,2010,201(10):1544-1550.
[12] Haanshuus CG,Mohn SC,M?rch K,et al. A novel,singleamplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen,Norway[J]. Malar J,2013,12(1):26.
[13] Sherman IW. Malaria:parasite biology,pathogenesis,and pro tection[M] Second edition. Washington D.C:American Society for Microbiology,1998:267-270.
[14]张玲玲,阮卫,陈华良,等.浙江省5例输入性卵形疟原虫wallikeri亚种感染病例的鉴定[J].中国寄生虫学与寄生虫病杂志,2014,32(5):361-365.
[15]陈梦妮,毛祥华,邓艳,等.云南省疟疾疫情病例疟原虫镜检疑难形态的鉴定[J].中国病原生物学杂志,2017,12(3):227-232.
[16]谭妍,李志峰,凌华,等. 2015年重庆市3例输入性卵形疟原虫wallikeri亚种感染病例的实验室诊断[J].热带病与寄生虫学,2016,14(3):141-144.
[17]李美,夏志贵,汤林华.卵形疟原虫wallikeri亚种及其基因检测体系的研究进展[J].中国寄生虫学与寄生虫病杂志,2014,32(1):64-67.
[18]贾西帅,周水茂,杨燕,等.武汉市两种输入性卵形疟巢式PCR检测[J].中国热带医学,2018,18(5):423-426.
[19]张玲玲,姚立农,陈华良,等.输入性卵形疟原虫细胞色素b、细胞色素c氧化酶Ⅰ和乳酸脱氢酶基因单核苷酸多态性分析[J].中国寄生虫学与寄生虫病杂志,2017,35(5):429-433
[20]李瑾,魏庆宽,徐超,等.山东省14例输入性卵形疟原虫wallikeri亚种的鉴定[J].中国人兽共患病学报,2018,34(11):991-995.