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Universal conventional and real-time PCR diagnosis tools for Sarcoptes scabiei.

Angelone-Alasaad S, Molinar Min A, Pasquetti M, Alagaili AN, D'Amelio S, Berrilli F, Obanda V, Gebely MA, Soriguer RC, Rossi L - Parasit Vectors (2015)

Bottom Line: The mite Sarcoptes scabiei has a known host-range of over 100 mammal species including humans.These methods, based on skin scrapings, were successfully used to etiologically confirm the diagnosis of different clinical degrees of sarcoptic mange in 48 animals belonging to six species.Two universal PCR-based diagnosis methods for S. scabiei were successfully designed and applied; one based on conventional end-point PCR and the other on TaqMan real-time PCR.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Veterinarie, Largo Paolo Braccini 2, 10095, Grugliasco, Italy. sameralasaad@hotmail.com.

ABSTRACT

Background: The mite Sarcoptes scabiei has a known host-range of over 100 mammal species including humans. One of the prime objectives of the Sarcoptes-World Molecular Network (WMN) is to design and develop universal Sarcoptes PCR-based diagnosis methods.

Methods: We describe here for the first time two universal mitochondrial-based diagnosis methods: (i) conventional end-point PCR and (ii) TaqMan real-time PCR. The design of both of these universal diagnosis methods was based on Sarcoptes samples collected from 23 host species in 14 countries.

Results: These methods, based on skin scrapings, were successfully used to etiologically confirm the diagnosis of different clinical degrees of sarcoptic mange in 48 animals belonging to six species. These universal PCR-based diagnosis methods are highly specific, technically sensitive and simple, and are based on the amplification of 135 bp from the Mitochondrial 16S rDNA. The method based on TaqMan real-time qPCR was more sensitive than the conventional end-point PCR.

Conclusions: Two universal PCR-based diagnosis methods for S. scabiei were successfully designed and applied; one based on conventional end-point PCR and the other on TaqMan real-time PCR. We recommend further testing and the application of these new universal methods worldwide.

No MeSH data available.


Related in: MedlinePlus

Universal normal end-point PCR amplification of mitochondrial DNA from Sarcoptes scabiei (in mangy wolves from Spain) at several dilutions, using the universal primers
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Fig2: Universal normal end-point PCR amplification of mitochondrial DNA from Sarcoptes scabiei (in mangy wolves from Spain) at several dilutions, using the universal primers

Mentions: The technical sensitivity of the end-point PCR diagnosis was lower than that of the TaqMan PCR diagnosis. The minimum amount of Sarcoptes gDNA detected with conventional end-point PCR was about 80 pg/μL (Fig. 2), whereas only 10 pg/μL was needed for the TaqMan PCR technique (Fig. 3). The higher sensitivity of the TaqMan real-time PCR diagnosis method was expected and can be attributed to the fact that the detection limit in a conventional end-point PCR is based on the final dilution at which a PCR product is still visible in agarose gels, while the fluorophore signal in the TaqMan probes is still detectable at much lower concentrations. The PCR mixtures/conditions of the TaqMan PCR and conventional end-point PCR may also have contributed to this difference.Fig. 2


Universal conventional and real-time PCR diagnosis tools for Sarcoptes scabiei.

Angelone-Alasaad S, Molinar Min A, Pasquetti M, Alagaili AN, D'Amelio S, Berrilli F, Obanda V, Gebely MA, Soriguer RC, Rossi L - Parasit Vectors (2015)

Universal normal end-point PCR amplification of mitochondrial DNA from Sarcoptes scabiei (in mangy wolves from Spain) at several dilutions, using the universal primers
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4644327&req=5

Fig2: Universal normal end-point PCR amplification of mitochondrial DNA from Sarcoptes scabiei (in mangy wolves from Spain) at several dilutions, using the universal primers
Mentions: The technical sensitivity of the end-point PCR diagnosis was lower than that of the TaqMan PCR diagnosis. The minimum amount of Sarcoptes gDNA detected with conventional end-point PCR was about 80 pg/μL (Fig. 2), whereas only 10 pg/μL was needed for the TaqMan PCR technique (Fig. 3). The higher sensitivity of the TaqMan real-time PCR diagnosis method was expected and can be attributed to the fact that the detection limit in a conventional end-point PCR is based on the final dilution at which a PCR product is still visible in agarose gels, while the fluorophore signal in the TaqMan probes is still detectable at much lower concentrations. The PCR mixtures/conditions of the TaqMan PCR and conventional end-point PCR may also have contributed to this difference.Fig. 2

Bottom Line: The mite Sarcoptes scabiei has a known host-range of over 100 mammal species including humans.These methods, based on skin scrapings, were successfully used to etiologically confirm the diagnosis of different clinical degrees of sarcoptic mange in 48 animals belonging to six species.Two universal PCR-based diagnosis methods for S. scabiei were successfully designed and applied; one based on conventional end-point PCR and the other on TaqMan real-time PCR.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Veterinarie, Largo Paolo Braccini 2, 10095, Grugliasco, Italy. sameralasaad@hotmail.com.

ABSTRACT

Background: The mite Sarcoptes scabiei has a known host-range of over 100 mammal species including humans. One of the prime objectives of the Sarcoptes-World Molecular Network (WMN) is to design and develop universal Sarcoptes PCR-based diagnosis methods.

Methods: We describe here for the first time two universal mitochondrial-based diagnosis methods: (i) conventional end-point PCR and (ii) TaqMan real-time PCR. The design of both of these universal diagnosis methods was based on Sarcoptes samples collected from 23 host species in 14 countries.

Results: These methods, based on skin scrapings, were successfully used to etiologically confirm the diagnosis of different clinical degrees of sarcoptic mange in 48 animals belonging to six species. These universal PCR-based diagnosis methods are highly specific, technically sensitive and simple, and are based on the amplification of 135 bp from the Mitochondrial 16S rDNA. The method based on TaqMan real-time qPCR was more sensitive than the conventional end-point PCR.

Conclusions: Two universal PCR-based diagnosis methods for S. scabiei were successfully designed and applied; one based on conventional end-point PCR and the other on TaqMan real-time PCR. We recommend further testing and the application of these new universal methods worldwide.

No MeSH data available.


Related in: MedlinePlus