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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

Specificity of the PI-LAMP.The micro-tubes containing the reaction mixture with propidium iodide have been exposed to UV after incubation. The positive reactions that returned an amplification product are evidenced by a bright fluorescence. The reaction have been performed by using purified DNA from larvae of 1) Acanthocheilonema reconditum; 2) Acanthocheilonema sp.; 3)Angiostrongylus vasorum; 4) Brugia sp.; 5) Cercopithifilaria sp.; 6) Dirofilaria immitis; 7) Loa; 8) Mansonella perstans; 9) Spirocerca lupi; 10) Wuchereria bancrofti; 12) and 13) Dirofilaria repens; 14) D. repens positive mosquito pool; 15) D. repens negative mosquito pool; 16) D. repens positive canine blood; 17) D. repens negative canine blood. The sample 11 is a negative sample with water instead of DNA.
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pntd.0004789.g003: Specificity of the PI-LAMP.The micro-tubes containing the reaction mixture with propidium iodide have been exposed to UV after incubation. The positive reactions that returned an amplification product are evidenced by a bright fluorescence. The reaction have been performed by using purified DNA from larvae of 1) Acanthocheilonema reconditum; 2) Acanthocheilonema sp.; 3)Angiostrongylus vasorum; 4) Brugia sp.; 5) Cercopithifilaria sp.; 6) Dirofilaria immitis; 7) Loa; 8) Mansonella perstans; 9) Spirocerca lupi; 10) Wuchereria bancrofti; 12) and 13) Dirofilaria repens; 14) D. repens positive mosquito pool; 15) D. repens negative mosquito pool; 16) D. repens positive canine blood; 17) D. repens negative canine blood. The sample 11 is a negative sample with water instead of DNA.

Mentions: However, when it was tested in vitro, no amplification was showed. Equally, no amplification was registered when DNAs from samples of D. immits, Ac. reconditum, An. vasorum, Brugia sp., L. loa, M. perstans or Cercopithifilaria sp. were used as template (able 2). On the contrary, all the ReT:LAMP reactions with DNA of D. repens returned the expected amplification curve (Fig 2a and 2b), and all the PI-LAMP mixtures were fluorescent (Fig 3).


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)

Specificity of the PI-LAMP.The micro-tubes containing the reaction mixture with propidium iodide have been exposed to UV after incubation. The positive reactions that returned an amplification product are evidenced by a bright fluorescence. The reaction have been performed by using purified DNA from larvae of 1) Acanthocheilonema reconditum; 2) Acanthocheilonema sp.; 3)Angiostrongylus vasorum; 4) Brugia sp.; 5) Cercopithifilaria sp.; 6) Dirofilaria immitis; 7) Loa; 8) Mansonella perstans; 9) Spirocerca lupi; 10) Wuchereria bancrofti; 12) and 13) Dirofilaria repens; 14) D. repens positive mosquito pool; 15) D. repens negative mosquito pool; 16) D. repens positive canine blood; 17) D. repens negative canine blood. The sample 11 is a negative sample with water instead of DNA.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004789.g003: Specificity of the PI-LAMP.The micro-tubes containing the reaction mixture with propidium iodide have been exposed to UV after incubation. The positive reactions that returned an amplification product are evidenced by a bright fluorescence. The reaction have been performed by using purified DNA from larvae of 1) Acanthocheilonema reconditum; 2) Acanthocheilonema sp.; 3)Angiostrongylus vasorum; 4) Brugia sp.; 5) Cercopithifilaria sp.; 6) Dirofilaria immitis; 7) Loa; 8) Mansonella perstans; 9) Spirocerca lupi; 10) Wuchereria bancrofti; 12) and 13) Dirofilaria repens; 14) D. repens positive mosquito pool; 15) D. repens negative mosquito pool; 16) D. repens positive canine blood; 17) D. repens negative canine blood. The sample 11 is a negative sample with water instead of DNA.
Mentions: However, when it was tested in vitro, no amplification was showed. Equally, no amplification was registered when DNAs from samples of D. immits, Ac. reconditum, An. vasorum, Brugia sp., L. loa, M. perstans or Cercopithifilaria sp. were used as template (able 2). On the contrary, all the ReT:LAMP reactions with DNA of D. repens returned the expected amplification curve (Fig 2a and 2b), and all the PI-LAMP mixtures were fluorescent (Fig 3).

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