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A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera).
J Invertebr Pathol. 2010 Oct; 105(2):151-5.JI

Abstract

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R(2)=0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.

Authors+Show Affiliations

School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20570679

Citation

Hamiduzzaman, Mollah Md, et al. "A Multiplex PCR Assay to Diagnose and Quantify Nosema Infections in Honey Bees (Apis Mellifera)." Journal of Invertebrate Pathology, vol. 105, no. 2, 2010, pp. 151-5.
Hamiduzzaman MM, Guzman-Novoa E, Goodwin PH. A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera). J Invertebr Pathol. 2010;105(2):151-5.
Hamiduzzaman, M. M., Guzman-Novoa, E., & Goodwin, P. H. (2010). A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera). Journal of Invertebrate Pathology, 105(2), 151-5. https://doi.org/10.1016/j.jip.2010.06.001
Hamiduzzaman MM, Guzman-Novoa E, Goodwin PH. A Multiplex PCR Assay to Diagnose and Quantify Nosema Infections in Honey Bees (Apis Mellifera). J Invertebr Pathol. 2010;105(2):151-5. PubMed PMID: 20570679.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera). AU - Hamiduzzaman,Mollah Md, AU - Guzman-Novoa,Ernesto, AU - Goodwin,Paul H, Y1 - 2010/06/04/ PY - 2010/04/14/received PY - 2010/05/27/revised PY - 2010/06/01/accepted PY - 2010/6/24/entrez PY - 2010/6/24/pubmed PY - 2011/1/20/medline SP - 151 EP - 5 JF - Journal of invertebrate pathology JO - J. Invertebr. Pathol. VL - 105 IS - 2 N2 - Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R(2)=0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available. SN - 1096-0805 UR - https://www.unboundmedicine.com/medline/citation/20570679/A_multiplex_PCR_assay_to_diagnose_and_quantify_Nosema_infections_in_honey_bees__Apis_mellifera__ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2011(10)00139-4 DB - PRIME DP - Unbound Medicine ER -