摘要
目的提高标签引物-套式/多重PCR诊断疟疾的敏感性、特异性与稳定性。方法用滤纸法采集非疟疾发热病人血样30份及其他传染病(感冒,流感,伤寒,肝炎等)患者血样20份;抽取恶性疟和间日疟各1例患者血4m1,进行系列稀释制备不同疟原虫含量的感染血样;健康者血样作对照。用热裂解法制备DNA模板,用线粒体细胞色素氧化酶基因作为靶基因,应用相关软件和网络资源(PrimerPremier5.0,PUBMED,NCBI-BLAST和Mfold服务器)设计和优化标签引物-套式/多重PCR,并用于检测所采集制备的各种血样。结果间日疟与恶性疟患者血系列稀释为含1000、100、10和1个虫/μl后经标签引物-套式/多重PCR检测,恶性疟和间日疟患者各稀释含虫血样分别在611bp和255bp出现1条带,能检测到原虫密度均为1个虫/μl血;非疟疾发热病人血样30份及其他传染病患者血样均为阴性;健康者血样未出现扩增条带,每种血样反复测试3次以上均获得同样结果。结论经优化的标签引物-套式/多重PCR在疟疾诊断中具有较高的敏感性、特异性和稳定性。
Objective To improve the sensitivity, specificity and stability of the Tag-primer nested/muhiplex PCR for malaria diagnosis. Methods Filter paper blood samples were collected from 30 non-malaria fever patients and 20 infectious disease patients （common cold, influenza, typhoid, hepatitis, etc.）. Four ml blood each taken from one falciparum malaria patient and one vivax malaria patient was serially diluted. Healthy blood sample was used as negative control. Improved direct heating method was used to prepare DNA template. The cytochrome oxidase gene （coxl） located in mitochondrion was selected as target gene. Relevant web resources and software （PUBMED, NCBI-BLAST, Mfold server and Primer Premier 5.0） were employed to design and optimize Tag-primer nested/multiplex PCR （UT-PCR） which was used to test all blood samples. Results A 611 bp band and a 255 bp band were seen in serially diluted infected blood samples （1 000, 100, 10 and 1 parasite/μl） from P.f and P.v patient tested by UT-PCR. The detection limit of either P.falciparum or P.vivax reached 1 parasite/μl, and the tested blood samples of non-malaria fever patients, patients with other infectious diseases and healthy persons were all negative. Consistent results of each sample in more than 3 duplicated tests were obtained. Conclusion The optimized Tag-primer nested/multiplex PCR shows high sensitivity, specificity and stability in malaria diagnosis.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2007年第3期213-216,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
广西科学研究与技术开发计划项目(No.0632007-3B)~~
