Validation of SYBR green I based closed‐tube loop‐mediated isothermal amplification (LAMP) assay for diagnosis of knowlesi malaria

Abstract

Background As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi. Methods A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes. Results LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2–99.7%) and 100% specific (95% CI 83.2–100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange. Conclusions These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.

Plasmodium knowlesi: the game changer for malaria eradication

“…To facilitate the end point detection of LAMP, a variety of DNA intercalating dyes [ 113 – 115 ], fluorescent indicator dyes [ 116 , 117 ] and pH indicators [ 118 ] can be included in the LAMP assay. Recently, Lai et al has developed a SYBR green I LAMP assay for the detection of P. knowlesi by targeting the 18s rRNA gene, where a positive reaction is indicated by green colour whereas the negative reaction is indicated by orange colour [ 119 ]. This new assay exhibited clinical sensitivity of 97.1% and clinical specificity of 100%.…”

Malaria: biochemical, physiological, diagnostic, and therapeutic updates

“…Given the advantages of minimum equipment, short time, 99% sensitivity and accurate diagnosis of asymptomatic malaria, LAMP is now considered a superior screening test than conventional methods for malaria diagnosis especially in non-endemic countries ( Feleke, Alemu & Yemanebirhane, 2021 ). Considering 18s rRNA as the target gene, SYBR green I also adds higher specificity to LAMP than the nested PCR ( Lai, Ooi & Lau, 2021 ).…”

Use of polypyrrole-polystyrene membranes for extracting DNA from plant tissues

“…The higher elution percentages obtained in the experiments using the Tris/SDS elution solution can be associated with the longer chains of the surfactant that favor the exchange with the phosphate groups of DNA. An interesting result of the DNA adsorption and elution process was shown in the fluorescence microscopy images after using the SYBR Green dye, which exhibits strong fluorescence after interaction with double-stranded nucleic acids [57] . From this, we could confirm the occurrence of the steps of capture and release of DNA from the membrane surface.…”