Development of a Loop-Mediated Isothermal Amplification (LAMP) for Diagnosis of Ascaris lumbricoides in Fecal Samples

Abstract

Ascaris lumbricoides is a nematode parasite that causes ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, require high expertise and limited by low sensitivity. This study sought to develop a loop-mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples. Primer Explorer V4 software was used to design primers based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA. Four consensus sets of primers, which successfully identified and amplified 6 regions of the target sequence, were adopted due to their reliability. Ascaris adult and ova were obtained from naturally infected school children, whose parents/guardians consented their participation in the study. Genomic DNA was extracted using HotShot alkaline lysis method after harvesting Ascaris ova from feces using the modified Wisconsin floatation method, and amplified by LAMP at 63oC for 45 minutes. Blinded samples were used as clinical samples to test for LAMP after direct DNA extraction using the QIAmp Fast DNA stool minikit. LAMP products were visualized by naked eyes on addition of SYBR green dye, a DNA intercalating orange dye that turns green if amplification is positive. This was additionally confirmed on agarose gel that produced ladder-like bands due to inverted repeats of the target sequence on the same strand. LAMP technique developed successfully and reliably detected Ascaris DNA from a single ovum and in fecal samples. The assay specifically detected Ascaris DNA without amplifying DNA from ova of hookworm, Trichuris trichiura and Schistosoma mansoni, parasites which commonly co-occur with A. lumbricoides in feces. The developed LAMP assay has great potential for use in ascariasis diagnosis at the point of care and in low infection intensity situation that are characterized by control and elimination campaigns.