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Application of RFLP-PCR-Based Identification for Sand Fly Surveillance in an Area Endemic for Kala-Azar in Mymensingh, Bangladesh.

Alam MS, Kato H, Fukushige M, Wagatsuma Y, Itoh M - J Parasitol Res (2012)

Bottom Line: Among 1,055 female sand flies successfully analyzed for the species identification individually, 64.4% flies was classified as Sergentomyia species, whereas 35.6% was identified as P. argentipes and no P. papatasi was found.Although infection of Leishmania within the sand flies was individually examined targeting leishmanial minicircle DNA, none of the 1,055 sand flies examined were positive for Leishmania infection.The RFLP-PCR could be useful tools for taxonomic identification and Leishmania infection monitoring in endemic areas of Bangladesh.

View Article: PubMed Central - PubMed

Affiliation: Parasitology Laboratory, International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh.

ABSTRACT
Mymensingh is the most endemic district for kala-azar in Bangladesh. Phlebotomus argentipes remains the only known vector although a number of sand fly species are prevalent in this area. Genotyping of sand flies distributed in a VL endemic area was developed by a PCR and restriction-fragment-length polymorphism (RFLP) of 18S rRNA gene of sand fly species. Using the RFLP-PCR analysis with AfaI and HinfI restriction enzymes, P. argentipes, P. papatasi, and Sergentomyia species could be identified. Among 1,055 female sand flies successfully analyzed for the species identification individually, 64.4% flies was classified as Sergentomyia species, whereas 35.6% was identified as P. argentipes and no P. papatasi was found. Although infection of Leishmania within the sand flies was individually examined targeting leishmanial minicircle DNA, none of the 1,055 sand flies examined were positive for Leishmania infection. The RFLP-PCR could be useful tools for taxonomic identification and Leishmania infection monitoring in endemic areas of Bangladesh.

No MeSH data available.


Related in: MedlinePlus

RFLP-PCR analyses of 18S rRNA genes of P. argentipes, two Sergentomyia species, and P. papatasi. PCR amplification with Lu.18S 1S and Lu.18S 1R primers was performed to amplify approximately 450 bp fragments of sand fly 18S rRNA gene. The PCR products were digested with HinfI, and the digested samples were separated by electrophoresis in a 3% agarose gel to produce DNA fragments. Lane M; 100-basepair ladder, lane 1; P. argentipes, lane 2; Sergentomyia species A, and lane 3; Sergentomyia species B. Lane 4; P. papatasi.
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fig2: RFLP-PCR analyses of 18S rRNA genes of P. argentipes, two Sergentomyia species, and P. papatasi. PCR amplification with Lu.18S 1S and Lu.18S 1R primers was performed to amplify approximately 450 bp fragments of sand fly 18S rRNA gene. The PCR products were digested with HinfI, and the digested samples were separated by electrophoresis in a 3% agarose gel to produce DNA fragments. Lane M; 100-basepair ladder, lane 1; P. argentipes, lane 2; Sergentomyia species A, and lane 3; Sergentomyia species B. Lane 4; P. papatasi.

Mentions: The RFLP patterns of morphologically identified P. argentipes and two Sergentomyia species, A and B, were analyzed on the 18S rRNA fragments of the 2,000 bp. The three species were clearly differentiated by single digestion with AfaI or HinfI restriction enzyme (Figures 1(a) and 1(b)). On the other hand, RFLP analysis of the 450 bp amplicons with HinfI differentiated P. argentipes, P. papatasi from Sergentomyia species, although two Sergentomyia species were indistinguishable (Figure 2). As preliminary study of the analysis showed that the 2,000 bp target was difficult to be amplified in part of the sand fly samples, the RFLP-PCR analysis of the 450 bp amplicon was employed for further genotyping of the samples from the field. The less efficiency for the amplification of longer fragments is probably associated with the postmortem changes that may occur in dead specimens in the light trap.


Application of RFLP-PCR-Based Identification for Sand Fly Surveillance in an Area Endemic for Kala-Azar in Mymensingh, Bangladesh.

Alam MS, Kato H, Fukushige M, Wagatsuma Y, Itoh M - J Parasitol Res (2012)

RFLP-PCR analyses of 18S rRNA genes of P. argentipes, two Sergentomyia species, and P. papatasi. PCR amplification with Lu.18S 1S and Lu.18S 1R primers was performed to amplify approximately 450 bp fragments of sand fly 18S rRNA gene. The PCR products were digested with HinfI, and the digested samples were separated by electrophoresis in a 3% agarose gel to produce DNA fragments. Lane M; 100-basepair ladder, lane 1; P. argentipes, lane 2; Sergentomyia species A, and lane 3; Sergentomyia species B. Lane 4; P. papatasi.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369511&req=5

fig2: RFLP-PCR analyses of 18S rRNA genes of P. argentipes, two Sergentomyia species, and P. papatasi. PCR amplification with Lu.18S 1S and Lu.18S 1R primers was performed to amplify approximately 450 bp fragments of sand fly 18S rRNA gene. The PCR products were digested with HinfI, and the digested samples were separated by electrophoresis in a 3% agarose gel to produce DNA fragments. Lane M; 100-basepair ladder, lane 1; P. argentipes, lane 2; Sergentomyia species A, and lane 3; Sergentomyia species B. Lane 4; P. papatasi.
Mentions: The RFLP patterns of morphologically identified P. argentipes and two Sergentomyia species, A and B, were analyzed on the 18S rRNA fragments of the 2,000 bp. The three species were clearly differentiated by single digestion with AfaI or HinfI restriction enzyme (Figures 1(a) and 1(b)). On the other hand, RFLP analysis of the 450 bp amplicons with HinfI differentiated P. argentipes, P. papatasi from Sergentomyia species, although two Sergentomyia species were indistinguishable (Figure 2). As preliminary study of the analysis showed that the 2,000 bp target was difficult to be amplified in part of the sand fly samples, the RFLP-PCR analysis of the 450 bp amplicon was employed for further genotyping of the samples from the field. The less efficiency for the amplification of longer fragments is probably associated with the postmortem changes that may occur in dead specimens in the light trap.

Bottom Line: Among 1,055 female sand flies successfully analyzed for the species identification individually, 64.4% flies was classified as Sergentomyia species, whereas 35.6% was identified as P. argentipes and no P. papatasi was found.Although infection of Leishmania within the sand flies was individually examined targeting leishmanial minicircle DNA, none of the 1,055 sand flies examined were positive for Leishmania infection.The RFLP-PCR could be useful tools for taxonomic identification and Leishmania infection monitoring in endemic areas of Bangladesh.

View Article: PubMed Central - PubMed

Affiliation: Parasitology Laboratory, International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh.

ABSTRACT
Mymensingh is the most endemic district for kala-azar in Bangladesh. Phlebotomus argentipes remains the only known vector although a number of sand fly species are prevalent in this area. Genotyping of sand flies distributed in a VL endemic area was developed by a PCR and restriction-fragment-length polymorphism (RFLP) of 18S rRNA gene of sand fly species. Using the RFLP-PCR analysis with AfaI and HinfI restriction enzymes, P. argentipes, P. papatasi, and Sergentomyia species could be identified. Among 1,055 female sand flies successfully analyzed for the species identification individually, 64.4% flies was classified as Sergentomyia species, whereas 35.6% was identified as P. argentipes and no P. papatasi was found. Although infection of Leishmania within the sand flies was individually examined targeting leishmanial minicircle DNA, none of the 1,055 sand flies examined were positive for Leishmania infection. The RFLP-PCR could be useful tools for taxonomic identification and Leishmania infection monitoring in endemic areas of Bangladesh.

No MeSH data available.


Related in: MedlinePlus