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 microscopybased 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.
