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Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Approach for the Rapid Detection of Dirofilaria repens from Biological Samples.

Raele DA, Pugliese N, Galante D, Latorre LM, Cafiero MA - PLoS Negl Trop Dis (2016)

Bottom Line: The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene.The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl).Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene.

View Article: PubMed Central - PubMed

Affiliation: Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Unit of Medical Entomology, Department of Virology, Foggia, Italy.

ABSTRACT
Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic.

No MeSH data available.


Related in: MedlinePlus

Results of the sensitivity assay.a) ReT-LAMP. The curves represent the ΔRn after subtraction of the ROX reference dye fluorescence. The amount of D. repens DNA for each reaction is indicated near the respective curve. *The curves of the reaction with 15 ag of DNA and of negative control are overlapping. b) PCR. M: AmpliSize Molecular Ruler, 50–2,000 bp Ladder (BioRad Laboratories, Milan, Italy). c) PI-LAMP. The bright fluorescence indicates the positivity of the reaction.
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pntd.0004789.g004: Results of the sensitivity assay.a) ReT-LAMP. The curves represent the ΔRn after subtraction of the ROX reference dye fluorescence. The amount of D. repens DNA for each reaction is indicated near the respective curve. *The curves of the reaction with 15 ag of DNA and of negative control are overlapping. b) PCR. M: AmpliSize Molecular Ruler, 50–2,000 bp Ladder (BioRad Laboratories, Milan, Italy). c) PI-LAMP. The bright fluorescence indicates the positivity of the reaction.

Mentions: The minimum amount of template necessary to obtain an amplification profile by the ReT LAMP was 0.15 fg, corresponding to about 50 copies of target (Fig 4a), while an amplicon was yielded by PCR (Fig 4b) from a minimum starting amount of 15 fg (about 5,000 copies of target).


Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Approach for the Rapid Detection of Dirofilaria repens from Biological Samples.

Raele DA, Pugliese N, Galante D, Latorre LM, Cafiero MA - PLoS Negl Trop Dis (2016)

Results of the sensitivity assay.a) ReT-LAMP. The curves represent the ΔRn after subtraction of the ROX reference dye fluorescence. The amount of D. repens DNA for each reaction is indicated near the respective curve. *The curves of the reaction with 15 ag of DNA and of negative control are overlapping. b) PCR. M: AmpliSize Molecular Ruler, 50–2,000 bp Ladder (BioRad Laboratories, Milan, Italy). c) PI-LAMP. The bright fluorescence indicates the positivity of the reaction.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920375&req=5

pntd.0004789.g004: Results of the sensitivity assay.a) ReT-LAMP. The curves represent the ΔRn after subtraction of the ROX reference dye fluorescence. The amount of D. repens DNA for each reaction is indicated near the respective curve. *The curves of the reaction with 15 ag of DNA and of negative control are overlapping. b) PCR. M: AmpliSize Molecular Ruler, 50–2,000 bp Ladder (BioRad Laboratories, Milan, Italy). c) PI-LAMP. The bright fluorescence indicates the positivity of the reaction.
Mentions: The minimum amount of template necessary to obtain an amplification profile by the ReT LAMP was 0.15 fg, corresponding to about 50 copies of target (Fig 4a), while an amplicon was yielded by PCR (Fig 4b) from a minimum starting amount of 15 fg (about 5,000 copies of target).

Bottom Line: The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene.The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl).Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene.

View Article: PubMed Central - PubMed

Affiliation: Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Unit of Medical Entomology, Department of Virology, Foggia, Italy.

ABSTRACT
Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic.

No MeSH data available.


Related in: MedlinePlus