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Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing.
Vet Parasitol 2008; 158(1-2):11-22VP

Abstract

Three LAMP (loop-mediated isothermal DNA amplification) assays were applied to detect Cryptosporidium species DNA in a total number of 270 fecal samples originating from cattle, sheep and horses in South Africa. DNA was extracted from 0.5 g of fecal material. Results of LAMP detection were compared to those obtained by nested PCR targeting the Cryptosporidium 18 small subunit rRNA (18S) gene. All samples were negative by nested PCR, while up to one-third of samples were positive by LAMP assays. The SAM-1 LAMP assay, shown to detect C. parvum, C. hominis and C. meleagridis, amplified Cryptosporidium DNA in 36 of 107 cattle (33.64%), in 26 of 85 sheep (30.5%) and in 17 of 78 horses (21.79%). The HSP LAMP specific to C. muris and C. andersoni, amplified Cryptosporidium DNA in one cow (0.9%), five sheep (5.8%) and seven horses (8.9%). The gp60 LAMP assay, shown to detect C. parvum produced no amplified Cryptosporidium DNA, likely due to low sample DNA concentrations. The specificity of LAMP assays was confirmed by sequencing of the LAMP products generated in positive samples. Sequence products from the three LAMP assays showed high identity to the target gene sequences confirming the specificity of LAMP. In this study, the LAMP procedure was clearly superior to nested PCR in the detection of Cryptosporidium species DNA. Use of LAMP is proposed as an efficient and effective tool for epidemiologic survey studies including screening of healthy animals in which Cryptosporidium oocyst shedding is characteristically low and likely below the detection limit of PCR in conventional sample concentrates.

Authors+Show Affiliations

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

18940521

Citation

Bakheit, Mohammed A., et al. "Sensitive and Specific Detection of Cryptosporidium Species in PCR-negative Samples By Loop-mediated Isothermal DNA Amplification and Confirmation of Generated LAMP Products By Sequencing." Veterinary Parasitology, vol. 158, no. 1-2, 2008, pp. 11-22.
Bakheit MA, Torra D, Palomino LA, et al. Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing. Vet Parasitol. 2008;158(1-2):11-22.
Bakheit, M. A., Torra, D., Palomino, L. A., Thekisoe, O. M., Mbati, P. A., Ongerth, J., & Karanis, P. (2008). Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing. Veterinary Parasitology, 158(1-2), pp. 11-22. doi:10.1016/j.vetpar.2008.09.012.
Bakheit MA, et al. Sensitive and Specific Detection of Cryptosporidium Species in PCR-negative Samples By Loop-mediated Isothermal DNA Amplification and Confirmation of Generated LAMP Products By Sequencing. Vet Parasitol. 2008 Nov 25;158(1-2):11-22. PubMed PMID: 18940521.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing. AU - Bakheit,Mohammed A, AU - Torra,Dena, AU - Palomino,Lily A, AU - Thekisoe,Oriel M M, AU - Mbati,Peter A, AU - Ongerth,Jerry, AU - Karanis,Panagiotis, Y1 - 2008/09/11/ PY - 2008/06/23/received PY - 2008/08/29/revised PY - 2008/09/03/accepted PY - 2008/10/23/pubmed PY - 2009/2/5/medline PY - 2008/10/23/entrez SP - 11 EP - 22 JF - Veterinary parasitology JO - Vet. Parasitol. VL - 158 IS - 1-2 N2 - Three LAMP (loop-mediated isothermal DNA amplification) assays were applied to detect Cryptosporidium species DNA in a total number of 270 fecal samples originating from cattle, sheep and horses in South Africa. DNA was extracted from 0.5 g of fecal material. Results of LAMP detection were compared to those obtained by nested PCR targeting the Cryptosporidium 18 small subunit rRNA (18S) gene. All samples were negative by nested PCR, while up to one-third of samples were positive by LAMP assays. The SAM-1 LAMP assay, shown to detect C. parvum, C. hominis and C. meleagridis, amplified Cryptosporidium DNA in 36 of 107 cattle (33.64%), in 26 of 85 sheep (30.5%) and in 17 of 78 horses (21.79%). The HSP LAMP specific to C. muris and C. andersoni, amplified Cryptosporidium DNA in one cow (0.9%), five sheep (5.8%) and seven horses (8.9%). The gp60 LAMP assay, shown to detect C. parvum produced no amplified Cryptosporidium DNA, likely due to low sample DNA concentrations. The specificity of LAMP assays was confirmed by sequencing of the LAMP products generated in positive samples. Sequence products from the three LAMP assays showed high identity to the target gene sequences confirming the specificity of LAMP. In this study, the LAMP procedure was clearly superior to nested PCR in the detection of Cryptosporidium species DNA. Use of LAMP is proposed as an efficient and effective tool for epidemiologic survey studies including screening of healthy animals in which Cryptosporidium oocyst shedding is characteristically low and likely below the detection limit of PCR in conventional sample concentrates. SN - 0304-4017 UR - https://www.unboundmedicine.com/medline/citation/18940521/Sensitive_and_specific_detection_of_Cryptosporidium_species_in_PCR_negative_samples_by_loop_mediated_isothermal_DNA_amplification_and_confirmation_of_generated_LAMP_products_by_sequencing_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0304-4017(08)00470-6 DB - PRIME DP - Unbound Medicine ER -