【摘要】：目的：建立一種檢測惡性瘧原蟲和間日瘧原蟲的SYBR Green實時PCR法,并將建立的方法進行初步臨床應(yīng)用。 方法：1.針對惡性瘧原蟲與間日瘧原蟲18S rRNA基因設(shè)計2對(3條)外引物和2對(3條)內(nèi)引物,建立可同時擴增出惡性瘧原蟲和間日瘧原蟲特異性片段的多重巢式PCR方法,并進行敏感性、特異性評價和臨床標(biāo)本檢測。2.利用上述巢式引物進行SYBR Green實時PCR反應(yīng),同時進行熔解曲線和Tm值分析。優(yōu)化實時PCR的反應(yīng)體系與反應(yīng)條件,檢測該方法的敏感性、特異性和重復(fù)性并進行臨床標(biāo)本檢測。 結(jié)果：1.多重巢式PCR可同時擴增出惡性瘧原蟲和間日瘧原蟲的18S rRNA基因片段長度分別為162bp(惡性瘧原蟲),112bp(間日瘧原蟲),并能檢出混合感染,該實驗檢測間日瘧原蟲的靈敏度為101copies/μl。特異性實驗結(jié)果顯示每對引物只檢測對應(yīng)的蟲種,54份臨床標(biāo)本的檢測結(jié)果與鏡檢法無差別(P0.05),敏感度為97.30%,特異度為5.88%。2. SYBR Green實時PCR構(gòu)建的熒光定量標(biāo)準(zhǔn)曲線循環(huán)閾值與模板濃度呈現(xiàn)良好的線性關(guān)系,擴增效率為101.884%,檢測靈敏度為101copies/μl,從提取DNA到完成檢測只需3小時,重復(fù)性好。該方法與三日瘧原蟲及卵形瘧原蟲無交叉反應(yīng),對臨床標(biāo)本的檢測結(jié)果與多重巢式PCR檢測結(jié)果一致。 結(jié)論：1.本研究建立的多重巢式PCR方法可同時檢測惡性瘧原蟲與間日瘧原蟲,敏感性高,特異性強,可用于基層部門對瘧疾的檢測與惡性瘧原蟲和間日瘧原蟲的鑒別。2.本研究建立的SYBR Green實時PCR方法具有污染風(fēng)險低、靈敏度高、不需熒光標(biāo)記探針、成本低及高通量等優(yōu)點,適用于瘧疾的檢測與惡性瘧原蟲和間日瘧原蟲的鑒別。圖11幅,表7個,參考文獻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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R531.3;R440

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1 汪圣強;周華云;李宗;劉耀寶;傅旭峰;朱京京;曹俊;高琪;;SYBR GreenⅠ染料法定量PCR用于人體瘧原蟲定量檢測及蟲種鑒別的研究[J];中國血吸蟲病防治雜志;2011年06期

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