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

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.