A comparative study of the use of standard and Pv-Rv primers in regular nest 2-PCR and touchdown nest 2-PCR to detect Plasmodium vivax

Malaria is a disease caused by Plasmodium infection. Of the 5 species that infect humans, Plasmodium vivax and Plasmodium falciparum are the two most common species found in Indonesia. Early and accurate diagnosis is crucial for effective treatment and disease management. Conventional diagnostic methods, microscopy, and rapid diagnostic tests (RDTs) have limitations in sensitivity and specificity. PCR-based assays have undergone significant enhancement such as the development of touchdown PCR and primer designing. However, the use of PCR-based assays in Plasmodium vivax infections still has several limitations including the formation of dimers and cross reactivity. Two isolated Plasmodium vivax DNA samples taken from malaria confirmed patients and stored in the Biomedical Laboratory, Faculty of Medicine, Universitas Brawijaya were tested with both standard Plasmodium vivax (rViv) primers and new (Pv-Rv) primer for Plasmodium vivax 18SSU RNA gene detection. Each pairs of primers underwent both regular nest 2-PCR and touchdown nest 2-PCR. The PCR products were checked by 3% agarose gel electrophoresis, and then quantified by using GelDoc Imaging System and ImageJ software. The paired t-test was used to analyze the results statistically. The results showed that, using rViv primers, there was no statistically significant difference in the ratio of band intensity to background intensity between regular nest 2-PCR and touchdown nest 2-PCR (P?> 0.05). However, when the background intensity was analyzed, a statistically significant difference was discovered (P < 0.05). By using Pv-Rv primers, a significant difference in the background intensity as well as in the ratio of band intensity to background intensity between regular nest 2-PCR and touchdown nest 2-PCR was observed (P < 0.05). The results indicated that application of touchdown nest 2-PCR had an effect in reducing background noise by using either rViv primers (standard primer) or Pv-Rv primers (new primer). Further research and modification of Pv-Rv primers were required for Plasmodium vivax identification.