Zhang Guo-Qing, Tang Lin-Hua, Guan Ya-Yi, Zhou Shui-Sen, Zheng Bin, Huang Fang, Wu Song, Liu Yan
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai 200025, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2007 Dec;25(6):451-6.
To develop a multiplex PCR protocol for amplification of five Plasmodium falciparum drug resistance related genes, thereby facilitate the rapid and high throughput analysis of the drug resistance molecular markers.
Five pairs of primers were designed according to the reference sequences by using Primer Premier 5.0 and Oligo 6.0 software. Drug resistance related genes, including P. falciparum chloroquine resistance transporter (Pfcrt), multi-drug resistance 1 (Pfmdr1), dihydropteroate synthetase (Pfdhps), dihydrofolate reductase (Pfdhfr) and sarco/endoplasmic reticulum Ca2+-ATPase (PfATPase6), were amplified by single-tube multiplex PCR using Hot Start Taq DNA Polymerase among negative controls (P. vivax, P. berghei, P. cynomolgi, Leishmania donovani, Cryptosporidium andersoni), blank control (using H2O as template), as well as P. falciparum laboratory isolates (3D7, Dd2, HB3, FCC1/HN and CMH/YN) and field samples (collected from Yunnan, Hainan of China and Myanmar). After amplification, the PCR products were analyzed by agarose gel electrophoresis. The sequencing results were aligned to the reference sequence using BLAST.
Five expected bands at 315, 437, 514, 594 and 770 bp were obtained with no additional or nonspecific products in P. falciparum laboratory isolates and field samples. The sequencing results were identical with the reference sequence except the polymorphism sites, and exhibited more than 98.5% homology. The multiplex amplification was performed successfully starting from 0.1 ng of DNA template. No band was observed in negative controls and blank control.
The present study establishes a method to amplify five Plasmodium falciparum drug resistance related genes harboring 21 SNPs by one-tube reaction. The multiplex PCR protocol showing high specificity and sensitivity is more convenient and efficient in analyzing the P. falciparum drug resistance molecular markers as compared with traditional nested PCR.
开发一种多重PCR方法，用于扩增5个恶性疟原虫耐药相关基因，从而促进耐药分子标志物的快速高通量分析。
使用Primer Premier 5.0和Oligo 6.0软件根据参考序列设计5对引物。采用热启动Taq DNA聚合酶，通过单管多重PCR扩增耐药相关基因，包括恶性疟原虫氯喹耐药转运蛋白（Pfcrt）、多药耐药1（Pfmdr1）、二氢蝶酸合酶（Pfdhps）、二氢叶酸还原酶（Pfdhfr）和肌浆/内质网Ca2+ -ATP酶（PfATPase6），阴性对照（间日疟原虫、伯氏疟原虫、食蟹猴疟原虫、杜氏利什曼原虫、安德逊隐孢子虫）、空白对照（以H2O为模板）以及恶性疟原虫实验室分离株（3D7、Dd2、HB3、FCC1/HN和CMH/YN）和现场样本（从中国云南、海南以及缅甸采集）。扩增后，通过琼脂糖凝胶电泳分析PCR产物。使用BLAST将测序结果与参考序列进行比对。
在恶性疟原虫实验室分离株和现场样本中获得了5条预期条带，大小分别为315、437、514、594和770 bp，无额外或非特异性产物。除多态性位点外，测序结果与参考序列一致，同源性超过98.5%。从0.1 ng DNA模板开始成功进行了多重扩增。在阴性对照和空白对照中未观察到条带。
本研究建立了一种通过单管反应扩增5个携带21个单核苷酸多态性的恶性疟原虫耐药相关基因的方法。与传统巢式PCR相比，该多重PCR方法具有高特异性和高灵敏度，在分析恶性疟原虫耐药分子标志物方面更方便、高效。
