Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2

SUMMARYSchistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.

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Optimization of duplex real-time PCR with melting-curve analysis for detecting the microsporidian parasites Nosema apis and Nosema ceranae in Apis mellifera

The Canadian Entomologist ◽

10.4039/n10-010 ◽

2010 ◽

Vol 142 (3) ◽

pp. 271-283 ◽

Cited By ~ 18

Author(s):

Karen L. Burgher-MacLellan ◽

Geoffrey R. Williams ◽

Dave Shutler ◽

Kenna MacKenzie ◽

Richard E.L. Rogers

Keyword(s):

Apis Mellifera ◽

Real Time ◽

Real Time Pcr ◽

Honey Bees ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Nosema Apis ◽

Conventional Pcr ◽

Pcr Assay

AbstractHoney bees, Apis mellifera (L.) (Hymenoptera: Apidae), are parasitized by the microsporidians Nosema apis (Zander) and Nosema ceranae (Fries). Molecular techniques are commonly used to differentiate between these parasites because light microscopy is inadequate. Our objectives were to (i) adapt the previously published duplex polymerase chain reaction (PCR) targeting the 16S rRNA gene of N. apis (321APIS-FOR, 321APIS-REV) and N. ceranae (218MITOC-FOR, 218MITOC-REV) using qualitative real-time PCR assay with SYBR® Green I dye (R-T PCR) and DNA melting-curve analysis, and (ii) determine whether the two Nosema species can be detected simultaneously in honey bees. Total spore counts and purified total genomic DNA were obtained from 37 bee samples (19 individual workers and 18 pooled samples of 15 workers) collected in Nova Scotia, Prince Edward Island, and Newfoundland, Canada. Overall, the prevalence of Nosema species was 86.5% (32/37 samples of bee DNA), based on conventional PCR and the optimized R-T PCR assay. The melting-curve analysis showed three groups of curve profiles that could determine the prevalence of N. apis, N. ceranae, and co-infection (21.9%, 56.2%, and 21.9%, respectively). The duplex R-T PCR assay was efficient, specific, and more sensitive than duplex conventional PCR because co-infection was identified in 5.4% (n = 2) more samples. Sequencing of R-T PCR products confirmed the results of the melting-curve analysis. Duplex R-T PCR with melting-curve analysis is a sensitive and rapid method of detecting N. apis, N. ceranae, and co-infection in honey bees.

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