Rapid detection of Dirofilaria immitis in mosquito vectors and dogs using a real-time fluorescence resonance energy transfer PCR and melting curve analysis

Thanchomnang, Tongjit; Intapan, Pewpan M.; Lulitanond, Viraphong; Sangmaneedet, Somboon; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Choochote, Wej; Maleewong, Wanchai

doi:10.1016/j.vetpar.2009.12.002

Cited by 19 publications

(10 citation statements)

References 14 publications

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“…Furthermore, this method offers a high throughput and is performed in a closed system, which eliminates the risk of poten- tial cross-over contamination because it does not require agarose gel electrophoresis to visualise the amplicons. The real-time FRET PCR-based method has been successfully used for the detection of several parasites, including Wuchereria bancrofti (Lulitanond et al 2004), Brugia malayi (Thanchomnang et al 2008(Thanchomnang et al , 2010a, Dirofilaria immitis (Thanchomnang et al 2010b), Toxoplasma gondii (Brenier-Pinchart et al 2007), Plasmodium falciparum (Ojurongbe et al 2007) and O. viverrini (Intapan et al 2008a(Intapan et al , b, 2009b.…”

Section: Discussionmentioning

confidence: 99%

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

Thanchomnang

Intapan

Sri-Aroon³

et al. 2011

Mem. Inst. Oswaldo Cruz

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Section: Discussionmentioning

confidence: 99%

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

Thanchomnang

Intapan

Sri-Aroon³

et al. 2011

Mem. Inst. Oswaldo Cruz

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“…togoi , and Ae. aegypti mosquitoes were artificially infected as previously described with W. bancrofti [12], B. malayi [20], and D. immitis [15], respectively.…”

Section: Methodsmentioning

confidence: 99%

“…However, these methods require analysis by agarose gel electrophoresis which is slow, having limited throughput and a bias toward carry-over contamination as well as spurious results. Recently, quantitative real-time PCR was reported to successfully detect W. bancrofti [12], B. malayi , B. pahangi [13,14], D. immitis [15], and Dirofilaria repens [16]. Also recently, a real-time PCR-based high-resolution melting (HRM) analysis was reported for detection and identification of B. malayi , B. pahangi , and D. immitis in blood samples in a single-tube assay [17].…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time P

et al. 2013

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A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

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“…is challenging and requires specialised parasitological skills, impairing a reliable, prompt diagnosis [10,25]. Over the past decades, several molecular PCR-based assays have been shown to provide rapid, sensitive, and species-specific methods for the detection and delineation of D. immitis and D. repens DNA in invertebrate hosts [22,23,26-30]. Some molecular tools have been applied especially for the entomological monitoring of human filariasis in endemic areas [24,31-33].…”

Section: Introductionmentioning

confidence: 99%

Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis

et al. 2012

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BackgroundDirofilaria immitis and Dirofilaria repens are transmitted by bloodsucking culicid mosquitoes belonging to Culex, Aedes, Ochlerotatus, Anopheles and Mansonia genera.The detection of filarioids in mosquitoes for assessing distribution of vectors and/or of pathogens in a given area (also known as “xenomonitoring”), when based on individual dissection of wild-caught female mosquitoes is time consuming and hardly applicable in large epidemiological surveys.Our study aimed to evaluate the recently developed duplex real-time PCR for screening large number of culicids and to assess their positivity for D. immitis and D. repens in an area where both species are endemic.MethodsA duplex real-time PCR was used to detect and differentiate D. immitis and D. repens in mosquitoes collected in six provinces of the Veneto region using 43 carbon dioxide-baited traps under the frame of an entomological surveillance program to monitor the vectors of West Nile disease. From early May till October 2010, unfed female mosquitoes (n = 40,892) were captured in 20 selected sites.ResultsMosquitoes identified as Culex pipiens, Ochlerotatus caspius, Aedes vexans and Culex modestus were grouped into 995 pools according to species, day and site of collection (from minimum of 1 to maximum of 57). Out of 955 pools, 23 (2.41 %) scored positive for Dirofilaria spp. of which, 21 (2.2 %) for D. immitis and two (0.21 %) for D. repens. An overall Estimated Rate of Infection (ERI) of 0.06 % was recorded, being higher in Och. caspius and Ae. vexans (i.e., 0.18 % and 0.14 %, respectively). At least one mosquito pool was positive for Dirofilaria spp. in each province with the highest ERI recorded in Vicenza and Padova provinces (i.e., 0.42% and 0.16 %, respectively). Mosquitoes collected in all provinces were positive for D. immitis whereas, only two (i.e., Padova and Rovigo) provinces scored positive for D. repens. All mosquito species, except for Cx. modestus, were positive for D. immitis, whereas D. repens was only found in Cx. pipiens.ConclusionsThe results suggest that both Dirofilaria species are endemic and may occur in sympatry in the examined area. The molecular approach herein used represents a powerful tool for surveillance programs of D. immitis and D. repens in the culicid vectors towards a better understanding of the epidemiology of the infections they cause and their seasonal transmission patterns.

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Rapid detection of Dirofilaria immitis in mosquito vectors and dogs using a real-time fluorescence resonance energy transfer PCR and melting curve analysis

Cited by 19 publications

References 14 publications

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time P

Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis

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