染料法荧光PCR鉴别卵形疟原虫curtisi和wallikeri亚种

Distinction of Plasmodium ovale curtisi and Plasmodium ovale wallikeri by using SYBR Green PCR

目的建立染料法荧光PCR快速鉴别卵形疟原虫curtisi和wallikeri亚种。方法比对卵形疟原虫curtisi和wallikeri亚种的网织红细胞结合蛋白2(porbp2)基因,设计特异性引物,建立扩增卵形疟原虫curtisi和wallikeri亚种的染料法荧光PCR方法,进行熔解曲线分析,测定各亚种扩增产物的熔解温度,进行特异性、重复性和准确性评价。结果熔解曲线分析结果显示,建立的方法扩增卵形疟原虫curtisi和wallikeri亚种的熔解温度相差明显,curtisi亚种为74.2℃,wallikeri亚种为75.7℃。该方法虽可检测到其他3种疟原虫,但敏感度较低,卵形疟与其他3种疟原虫的熔解温度差别明显。重复性实验表明,该方法扩增卵形疟原虫curtisi和wallikeri亚种熔解温度变异系数均低于1%,重复性良好。对27份卵形疟原虫阳性样本的检测结果与巢式PCR分型结果完全一致。结论建立的染料法荧光PCR方法可快速对两种亚型卵形疟原虫进行鉴别。

Objective To develop a SYBR Green PCR method for Plasmodium ovale curtisi(Poc) and Plasmodium ovale wallikeri(Pow) identification. Methods According to the sequence of Plasmodium ovale reticulocyte-binding protein homologue 2(Porbp2) gene,the primer set was designed based on the both highly conserved and variable regions. Melting curve analysis was conducted following SYBR Green PCR for two species of Plasmodium ovale identification.The specificity,reproducibility and veracity of the method were validated. Results The melting temperatures(Tm) of Poc and Pow amplification were significantly different,being 74.2℃,75.7℃ respectively. The sequence of P. falciparum,P. malariae and P. vivax could be amplified,with poor sensitivity,and the Tm value was distinct with that of P. ovale. The CV values of Tm of Poc and Pow amplification were all less than 1%,showed qualified reproducibility.The method was compared with nest-PCR method to detect 27 P. ovale samples,the coincidence rate was 100%.Conclusion The SYBR Green PCR method developed in this study can be used for two distinct species of Plasmodium ovale identificaton.