中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2008年
5期
275-278
,共4页
疟原虫,恶性%染色与标记%DNA%聚合酶链反应
瘧原蟲,噁性%染色與標記%DNA%聚閤酶鏈反應
학원충,악성%염색여표기%DNA%취합매련반응
Plasmodium falciparum%Staining and labeling%DNA%Polymerase chain reaction
目的 建立从恶性疟原虫薄血涂片中提取DNA进行恶性疟原虫18S RNA基因套式PCR检测方法 ,并探讨其可行性.方法 利用螯合型的离子交换树脂Chelex-100作为介质,一步法分别提取患者染色和未染色薄血涂片恶性疟原虫DNA,行套式PCR扩增.以培养的不同浓度恶性疟原虫制备的染色和未染色薄血涂片提取恶性疟原虫DNA为模板进行PCR扩增,检测该方法的灵敏度.结果 用Chelex-100法提取恶性疟原虫患者染色与未染色薄血膜DNA,PCR扩增均出现205 bp特异扩增片段.染色及未染色薄血膜恶性疟原虫PCR扩增的最低虫体浓度分别为1.5×101/μL血和1.5×10-1/μL血.结论 Chelex-100法薄血涂片中提取微量DNA的套式PCR检测方法,可在基因水平检测存档的薄血涂片标本.为恶性疟疾的临床实验室诊断和分子流行病学研究提供了新方法.
目的 建立從噁性瘧原蟲薄血塗片中提取DNA進行噁性瘧原蟲18S RNA基因套式PCR檢測方法 ,併探討其可行性.方法 利用螯閤型的離子交換樹脂Chelex-100作為介質,一步法分彆提取患者染色和未染色薄血塗片噁性瘧原蟲DNA,行套式PCR擴增.以培養的不同濃度噁性瘧原蟲製備的染色和未染色薄血塗片提取噁性瘧原蟲DNA為模闆進行PCR擴增,檢測該方法的靈敏度.結果 用Chelex-100法提取噁性瘧原蟲患者染色與未染色薄血膜DNA,PCR擴增均齣現205 bp特異擴增片段.染色及未染色薄血膜噁性瘧原蟲PCR擴增的最低蟲體濃度分彆為1.5×101/μL血和1.5×10-1/μL血.結論 Chelex-100法薄血塗片中提取微量DNA的套式PCR檢測方法,可在基因水平檢測存檔的薄血塗片標本.為噁性瘧疾的臨床實驗室診斷和分子流行病學研究提供瞭新方法.
목적 건립종악성학원충박혈도편중제취DNA진행악성학원충18S RNA기인투식PCR검측방법 ,병탐토기가행성.방법 이용오합형적리자교환수지Chelex-100작위개질,일보법분별제취환자염색화미염색박혈도편악성학원충DNA,행투식PCR확증.이배양적불동농도악성학원충제비적염색화미염색박혈도편제취악성학원충DNA위모판진행PCR확증,검측해방법적령민도.결과 용Chelex-100법제취악성학원충환자염색여미염색박혈막DNA,PCR확증균출현205 bp특이확증편단.염색급미염색박혈막악성학원충PCR확증적최저충체농도분별위1.5×101/μL혈화1.5×10-1/μL혈.결론 Chelex-100법박혈도편중제취미량DNA적투식PCR검측방법,가재기인수평검측존당적박혈도편표본.위악성학질적림상실험실진단화분자류행병학연구제공료신방법.
Objective To investigate the feasibility of Chelex DNA extraction from thin blood smears for genetic analysis, and to develop smear-based nested polymerase chain reaction (PCR) of the 18S RNA of Plasrnodium falciparum. Methods Chelex-100 which was chelating ion exchange resin was used to extract DNA from Giemsa-stained or unstained thin blood smears of different concentrations of Plasmodium falciparum. With the extracted DNA as the template, 18S RNA gene was amplified by nested PCR to test the susceptibility of Chelex method. Results Positive band of 205bp appeared in nested PCR with DNA extracted from Giemsa-stained or unstained thin blood smears of patient with falciparum malaria. Using the Chelex method, the detection limits of the smear-based nested PCR were 1.5 × 101 parasite/μL blood for Giemsa-stained and 1.5×10-1 parasite/μL blood for unstained thin blood smears. Conclusions Chelex DNA extraction is a simple and efficient method for extracting trace amount of DNA from thin blood smear. The smear-based nested PCR developed in this study is feasible to identify the gene from reserved thin blood smears and will provide a new approach for clinical diagnosis and study of molecular epidemiology.
