Development of Quantitative Polymerase Chain (qPCR) Method Using 18S rRNA Gene as a Standard to Detect Plasmodium falciparum Infection Quantitatively in Indonesia

• Main Author: Syafira, Intan
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/42200
• Institution: Institut Teknologi Bandung

Plasmodium falciparum infection dominate approximately 62 percent of malaria total cases in Indonesia. As malaria endemic country, asymptomatic carriers remain a challenge for malaria elimination agenda in Indonesia. Being more sensitive than microscopy and rapid diagnostic test, quantitative polymerase chain reaction (qPCR) is more frequently used to detect asymtomatic infection. 18S rRNA gene is widely used as standard in qPCR method for its copies number in Plasmodium falciparum cell and its variable region in every species allowing for species identification. Despite its status as malaria endemic country, Indonesia still relies on qPCR-based diagnostic kits developed abroad, thus it is urgently needed to develop Indonesia’s own qPCR to be used independently. Therefore, the aim of this research is to develop qPCR method using 18S rRNA gene of Plasmodium falciparum as a standard to quantify its infection By aligning nine 18S rRNA genes of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale, a 105-base-pair of Pf18S rRNA gene is decided as standard to plot standard curve for Plasmodium falciparum infection quantification. Standard curve is plotted in SYBR Green assay using Plasmid PUC57 inserted with a 105-base-pair Pf18S rRNA gene with nine concentration variations. From 1 ng/?L to 10-8 ng/?L. Then, the standard curve is tested with three field samples, named as A219, B202, and B204. The standard curve equation is y = -3.4497x + 37.476 with R2 value and amplification efficiency is 0.9887 and 94.93%, respectively. On the assumption that a parasite equals to three copies number of Pf18S rRNA gene, the standard curve can quantify as little as 1.1 parasites/?L. Parasites concentration of filed samples B204 and B202 is 1.12 x 106 and 8 x 105 parasites/?L, respectively. To sum up, obtained in this research are qPCR condition and a pair of primer that can amplify a 105-base-pair of Pf18S rRNA gene used as a standard in qPCR method.