间日疟原虫孢子期SSUrRNA基因扩增、鉴定及其诊断应用

目的体外扩增、克隆间日疟原虫孢子期SSUrRNA编码基因特异性片段，分析其分子特征，评价其核酸诊断应用效果。方法根据间日疟原虫基因库相关核酸序列设计引物，采用聚合酶链反应技术从间日疟患者血样DNA提取物中扩增出间日疟原虫SSUrRNA基因片段，与pGEM—Teasy质粒连接构建重组子并转化大肠杆菌JM109；阳性克隆以双酶切鉴定后，双脱氧末端终止法测定分析其序列特征；以SSUrDNA为靶基因，建立间日疟PCR诊断方法，并以镜检法为标准评价其用于间日疟原虫感染的检测效果。蛄果从间日疟患者血样中扩增的SSUrDNA片段大小约为267bp；阳性克隆双酶切及PCR扩增均得到预期大小的片段；核酸序列测定显示插入的SSUrRNA基因扩增片段，含有267个核苷酸，与间日疟原虫Sall株孢子期SSUrRNA基因序列比较，同源性为99．3％，其中第220住为插入了1个碱基“T”，243住碱基由“G”取代了“T”。将纯化的含有SSUrRNA靶基因的重组质粒以正常人血液核酸提取液倍比稀释成浓度梯度作模板，PER扩增结果显示检测灵敏度至少迭102copy／μl；特异性检验显示仅间日疟原虫患者血样扩增出约267bp的特异性基因片段，而恶性疟原虫、弓形虫、血吸虫、肝吸虫感染患者血样及正常人血未见特异性扩增条带。以镜检法为参照标准，PCR敏感性为100％（102／102），特异性为100％（76／76）。结论所扩增克隆的间日疟原虫孢子期SSUrDNA片段序列具有种特异性且相对稳定，不同地理株间存在单核苷酸多态性，以其为靶基因建立的PCR检测方法敏感、特异，具有良好的推广使用价值。

Amplification of specific SSUrRNA gene sequence and its application in diagnosis of Plasmodium vivax malaria

Objective To amplified and analyze the specific sequence of sporozoite stage SSUrRNA gene fragment of P. vivax isolate, and use it as target to diagnose P. vivax malaria by polymerase chain reaction (PCR). Methods The SSUrDNA fragment was amplified from the DNA extracts of aN infected patient blood sample from Shenzhen in China using PCR method, which was ligated with plasmid pGEM-Teasy and transformed into E. coli JM109. Positive bacteria clones were identified by PCR and double enzyme digestion methods. The sequence of inserted SSUrDNA fragment was also determined and analyzed. Based on the SSUrRNA gene sequence as target, a PCR method was established for detection of P. vivax in blood samples and compared with microscopy examination. Results The amplified SSUrDNA fragment was about 267 bp in length, as aligned with P. vivax Sal 1 isolate the identity of nucleotides was 99.3%. Using thick blood film microscopy as gold standard, the sensitivity and specificity of the established PCR method were 100% (102/102, 76/76). Conclusion The amplified P.vivax sprorozoite stage SSUrRNA gene fragment sequence is relatively conserved in different strains, the PCR based method using the sequence as target to detect P. vivax in blood samples is specific and sensitive.