This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features!
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log in
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016:2016:7376207.
doi: 10.1155/2016/7376207. Epub 2016 Nov 1.

Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Fecal Samples

Affiliations

Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Fecal Samples

Esther A Shiraho et al. J Parasitol Res. 2016.

Abstract

Ascaris lumbricoides is a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA. We used Primer Explorer V4 software to design primers. Ascaris adult and ova were obtained from naturally infected school children, whose parents/guardians gave consent for their participation in the study. Genomic DNA was extracted using alkaline lysis method and amplified by LAMP at 63°C for 45 minutes. LAMP products were visualized by naked eyes after adding SYBR Green dye and also on agarose gel. LAMP successfully and reliably detected Ascaris DNA from a single egg and in fecal samples. The assay specifically detected Ascaris DNA without amplifying DNA from ova of other parasites which commonly coexist 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 characterize control and elimination campaigns.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The various ratio of inner : outer primers tested. The 6 : 1 ratio readily amplified DNA of adult A. lumbricoides worm (positive control).
Figure 2
Figure 2
(a) Shows the sensitivity of LAMP visualized by 2% agarose gel electrophoresis. Lanes 1, 2, 3, 4, 5, and 6 are DNA extracted from 1, 5, 10, 15, 20, and 25 eggs, respectively. Lane M is the 100 bp molecular marker and N is the negative control. (b) LAMP visual detection for color change using the SYBR Green dye. Tubes 1, 2, 3, 4, 5, and 6 represent DNA extracted from 1, 5, 10, 15, 20, and 25 eggs, respectively, through 6. N is negative control.
Figure 3
Figure 3
(a) Specificity of LAMP assay by gel electrophoresis. Lane 1, A. lumbricoides adult worm; lane 2, Ascaris ova DNA; lane 3, hookworm ova DNA; lane 4, S. mansoni ova DNA; lane 5, T. trichiura ova DNA; lane N, negative control; and M = 100 bp molecular ruler (Thermo Scientific). (b) Visual detection by color change using the SYBR Green dye. Tube 1, A. lumbricoides adult worm; tube 2, Ascaris ova DNA; tube 3, hookworm ova DNA; tube 4, S. mansoni ova DNA; tube 5, T. trichiura ova DNA; and lane N, negative control.

References

    1. Hotez P. J., Kamath A. Neglected tropical diseases in sub-Saharan Africa: review of their prevalence, distribution, and disease burden. PLoS Neglected Tropical Diseases. 2009;3(8, article e412) doi: 10.1371/journal.pntd.0000412. - DOI - PMC - PubMed
    1. de Silva N. R., Brooker S., Hotez P. J., Montresor A., Engels D., Savioli L. Soil-transmitted helminth infections: updating the global picture. Trends in Parasitology. 2003;19(12):547–551. doi: 10.1016/j.pt.200310002. - DOI - PubMed
    1. Bethony J., Brooker S., Albonico M., et al. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. The Lancet. 2006;367(9521):1521–1532. doi: 10.1016/s0140-6736(06)68653-4. - DOI - PubMed
    1. Dangana A., Abayomi R. O., Way G. D., Akobi O. A. Survey of Ascaris lumbricoides among pupils of primary school in Jos south local government area of Plateau State, Nigeria. African Journal of Microbiology Research. 2011;5(17):2524–2527.
    1. Gordon C. A., Gray D. J., Gobert G. N., McManus D. P. DNA amplification approaches for the diagnosis of key parasitic helminth infections of humans. Molecular and Cellular Probes. 2011;25(4):143–152. doi: 10.1016/j.mcp.201105002. - DOI - PubMed
Cite

AltStyle によって変換されたページ (->オリジナル) /