Limits...
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

Relative positions of LAMP primers within the cytochrome c oxidase gene of D. repens.The first steps of the LAMP reaction are also depicted. i) Primers F3/B3, and DIRF2/DIRB2 (parts of DIRFIP and DIRBIP, respectively), recognize and anneal to their respective complementary targets. The DNA synthesis starts and the strand displacement activity of the DNA polymerase let the strand synthesized from DIRF2 and DIRB2 to be detached from the template strand. ii) The displaced neo-synthesized strands bring, at their termini, DIRF1c and DIRB1c that iii) will self-anneal to the DIRF1 and DIRB1 loci, respectively, thus forming a secondary structure with a loop. DIRB3/DIRF3 and DIRBIP/DIRFIP (specifically with the portions DIRB2 and DIRF2) hybridize with the complementary regions of the strand. The synthesis and the displacement of strand occur again, catalyzed by the same DNA polymerase. iv) the 3’ and 5’ termini, bearing DIRF1/DIRB1 and DIRB1c/DIRF1c, respectively, self-anneal to the complementary loci harbored by the same strand, which, in turn, will form the typical dumbbell structure. The DNA polymerase will catalyze the DNA synthesis starting from the 3’ terminus, displacing the 5’ terminus, and producing a concatamer which will be the basis for following the amplification steps.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4920375&req=5

pntd.0004789.g001: Relative positions of LAMP primers within the cytochrome c oxidase gene of D. repens.The first steps of the LAMP reaction are also depicted. i) Primers F3/B3, and DIRF2/DIRB2 (parts of DIRFIP and DIRBIP, respectively), recognize and anneal to their respective complementary targets. The DNA synthesis starts and the strand displacement activity of the DNA polymerase let the strand synthesized from DIRF2 and DIRB2 to be detached from the template strand. ii) The displaced neo-synthesized strands bring, at their termini, DIRF1c and DIRB1c that iii) will self-anneal to the DIRF1 and DIRB1 loci, respectively, thus forming a secondary structure with a loop. DIRB3/DIRF3 and DIRBIP/DIRFIP (specifically with the portions DIRB2 and DIRF2) hybridize with the complementary regions of the strand. The synthesis and the displacement of strand occur again, catalyzed by the same DNA polymerase. iv) the 3’ and 5’ termini, bearing DIRF1/DIRB1 and DIRB1c/DIRF1c, respectively, self-anneal to the complementary loci harbored by the same strand, which, in turn, will form the typical dumbbell structure. The DNA polymerase will catalyze the DNA synthesis starting from the 3’ terminus, displacing the 5’ terminus, and producing a concatamer which will be the basis for following the amplification steps.

Mentions: The available sequences (Accession numbers AJ271614, AM749230, AM749231, AM749232, AM749233, AM749234, DQ358814, JF461458 and KF692102) of D. repens cytochrome oxidase subunit I (COI) from GenBank were aligned by ClustalW, implemented in MEGA 6.0 [27]. The resulting consensus sequence was then used to design LAMP primers by the mean of the Primer Explorer Program (Available online at http://primerexplorer.jp/e/, latest accessed 8th April 2016). The primers are listed in Table 1. Because no complete sequences of D. repens COI are currently available, the relative positions of oligonucleotides are shown in Fig 1.


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)

Relative positions of LAMP primers within the cytochrome c oxidase gene of D. repens.The first steps of the LAMP reaction are also depicted. i) Primers F3/B3, and DIRF2/DIRB2 (parts of DIRFIP and DIRBIP, respectively), recognize and anneal to their respective complementary targets. The DNA synthesis starts and the strand displacement activity of the DNA polymerase let the strand synthesized from DIRF2 and DIRB2 to be detached from the template strand. ii) The displaced neo-synthesized strands bring, at their termini, DIRF1c and DIRB1c that iii) will self-anneal to the DIRF1 and DIRB1 loci, respectively, thus forming a secondary structure with a loop. DIRB3/DIRF3 and DIRBIP/DIRFIP (specifically with the portions DIRB2 and DIRF2) hybridize with the complementary regions of the strand. The synthesis and the displacement of strand occur again, catalyzed by the same DNA polymerase. iv) the 3’ and 5’ termini, bearing DIRF1/DIRB1 and DIRB1c/DIRF1c, respectively, self-anneal to the complementary loci harbored by the same strand, which, in turn, will form the typical dumbbell structure. The DNA polymerase will catalyze the DNA synthesis starting from the 3’ terminus, displacing the 5’ terminus, and producing a concatamer which will be the basis for following the amplification steps.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004789.g001: Relative positions of LAMP primers within the cytochrome c oxidase gene of D. repens.The first steps of the LAMP reaction are also depicted. i) Primers F3/B3, and DIRF2/DIRB2 (parts of DIRFIP and DIRBIP, respectively), recognize and anneal to their respective complementary targets. The DNA synthesis starts and the strand displacement activity of the DNA polymerase let the strand synthesized from DIRF2 and DIRB2 to be detached from the template strand. ii) The displaced neo-synthesized strands bring, at their termini, DIRF1c and DIRB1c that iii) will self-anneal to the DIRF1 and DIRB1 loci, respectively, thus forming a secondary structure with a loop. DIRB3/DIRF3 and DIRBIP/DIRFIP (specifically with the portions DIRB2 and DIRF2) hybridize with the complementary regions of the strand. The synthesis and the displacement of strand occur again, catalyzed by the same DNA polymerase. iv) the 3’ and 5’ termini, bearing DIRF1/DIRB1 and DIRB1c/DIRF1c, respectively, self-anneal to the complementary loci harbored by the same strand, which, in turn, will form the typical dumbbell structure. The DNA polymerase will catalyze the DNA synthesis starting from the 3’ terminus, displacing the 5’ terminus, and producing a concatamer which will be the basis for following the amplification steps.
Mentions: The available sequences (Accession numbers AJ271614, AM749230, AM749231, AM749232, AM749233, AM749234, DQ358814, JF461458 and KF692102) of D. repens cytochrome oxidase subunit I (COI) from GenBank were aligned by ClustalW, implemented in MEGA 6.0 [27]. The resulting consensus sequence was then used to design LAMP primers by the mean of the Primer Explorer Program (Available online at http://primerexplorer.jp/e/, latest accessed 8th April 2016). The primers are listed in Table 1. Because no complete sequences of D. repens COI are currently available, the relative positions of oligonucleotides are shown in Fig 1.

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