SYBR Green实时PCR检测恶性疟原虫与间日疟原虫方法的建立

发布时间：2019-05-18 09:10

【摘要】：目的：建立一种检测恶性疟原虫和间日疟原虫的SYBR Green实时PCR法,并将建立的方法进行初步临床应用。方法：1.针对恶性疟原虫与间日疟原虫18S rRNA基因设计2对(3条)外引物和2对(3条)内引物,建立可同时扩增出恶性疟原虫和间日疟原虫特异性片段的多重巢式PCR方法,并进行敏感性、特异性评价和临床标本检测。2.利用上述巢式引物进行SYBR Green实时PCR反应,同时进行熔解曲线和Tm值分析。优化实时PCR的反应体系与反应条件,检测该方法的敏感性、特异性和重复性并进行临床标本检测。结果：1.多重巢式PCR可同时扩增出恶性疟原虫和间日疟原虫的18S rRNA基因片段长度分别为162bp(恶性疟原虫),112bp(间日疟原虫),并能检出混合感染,该实验检测间日疟原虫的灵敏度为101copies/μl。特异性实验结果显示每对引物只检测对应的虫种,54份临床标本的检测结果与镜检法无差别(P0.05),敏感度为97.30%,特异度为5.88%。2. SYBR Green实时PCR构建的荧光定量标准曲线循环阈值与模板浓度呈现良好的线性关系,扩增效率为101.884%,检测灵敏度为101copies/μl,从提取DNA到完成检测只需3小时,重复性好。该方法与三日疟原虫及卵形疟原虫无交叉反应,对临床标本的检测结果与多重巢式PCR检测结果一致。结论：1.本研究建立的多重巢式PCR方法可同时检测恶性疟原虫与间日疟原虫,敏感性高,特异性强,可用于基层部门对疟疾的检测与恶性疟原虫和间日疟原虫的鉴别。2.本研究建立的SYBR Green实时PCR方法具有污染风险低、灵敏度高、不需荧光标记探针、成本低及高通量等优点,适用于疟疾的检测与恶性疟原虫和间日疟原虫的鉴别。图11幅,表7个,参考文献53篇。
[Abstract]:Objective: to establish a SYBR Green real-time PCR method for the detection of Plasmodium falciparum and Plasmodium vivax. Methods: 1. Two pairs of external primers and two pairs of internal primers were designed for the 18s rRNA gene of Plasmodium falciparum and Plasmodium vivax, and a multiplex PCR method was established to amplify the specific fragments of Plasmodium falciparum and Plasmodium vivax at the same time. Sensitivity, specificity evaluation and clinical specimen detection were carried out. 2. The SYBR Green real-time PCR reaction was carried out with the above nest primers, and the melting curve and TM value were analyzed at the same time. The reaction system and reaction conditions of real-time PCR were optimized, the sensitivity, specificity and reproducibility of the method were detected, and the clinical specimens were detected. Results: 1. The 18s rRNA gene fragments of Plasmodium falciparum and Plasmodium vivax could be amplified by multiplex PCR to 162bp (Plasmodium falciparum) and 112bp (Plasmodium vivax), respectively, and the mixed infection could be detected. The sensitivity of this experiment for the detection of Plasmodium vivax is 101copies/ 渭 l. The results of specific experiment showed that only the corresponding insect species were detected by each pair of primers. The results of 54 clinical specimens were not different from those of microscopic examination (P 0.05). The sensitivity and specificity of 54 clinical specimens were 97.30% and 5.88% respectively. The cycle threshold of fluorescence quantitative standard curve constructed by SYBR Green real-time PCR has a good linear relationship with template concentration, the amplification efficiency is 101.884%, the detection sensitivity is 101copies/ 渭 l, and it takes only 3 hours from DNA extraction to completion of detection, and the reproducibility is good. There was no cross reaction with Plasmodium falciparum and Plasmodium ovale, and the results of clinical specimens were consistent with those of multiplex nest PCR. Conclusion: 1. The multiplex PCR method established in this study can detect Plasmodium falciparum and Plasmodium vivax at the same time. It has high sensitivity and specificity, and can be used for the detection of malaria in grass-roots departments and the identification of Plasmodium falciparum and Plasmodium vivax. 2. The SYBR Green real-time PCR method established in this study has the advantages of low pollution risk, high sensitivity, no need for fluorescence labeling probe, low cost and high throughput. It is suitable for malaria detection and identification of Plasmodium falciparum and Plasmodium vivax. Fig. 11, table 7, references 53.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R531.3;R440

【参考文献】