Differential detection of Brugia malayi and Brugia pahangi by real-time fluorescence resonance energy transfer PCR and its evaluation for diagnosis of B. pahangi-infected dogs
- 182 Downloads
A real-time fluorescence resonance energy transfer PCR combined with melting curve analysis was developed for differentiating Brugia malayi and Brugia pahangi DNA in host blood using one set of primers and fluorophore-labeled hybridization probes specific for HhaI repetitive DNA. The differentiation of both species was based on their melting temperatures (Tm). The mean Tm ± SD of B. malayi and B. pahangi were 56.18 ± 0.21 and 52.49 ± 0.07, respectively. The method was used for the molecular detection of B. pahangi in infected dog blood samples. The diagnostic sensitivity, specificity, accuracy, and positive and negative predictive values of this method were 100%. The detected mean difference of the Tm might allow the simple discrimination of two related species. This method is fast, sensitive, allows for a high throughput, can be performed on very small volumes, and has potential for diagnosis of B. pahangi-infected dogs in endemic areas as well as for large epidemiological investigations.
KeywordsFluorescence Resonance Energy Transfer Melting Curve Analysis Babesia Differential Detection Restriction Fragment Length Polymorphism Profile
This study was supported by grants from the Office of the Higher Education Commission (grant funded under the Strategic Scholarships for Frontier Research Networks), the Thailand Research Fund, and the Research and Technology Transfer Affairs and the Faculty of Medicine (research grant Comm51201), Khon Kaen University, Thailand. The authors wish to thank Wej Choochote for his technical support. All investigations in this study complied with current Thai laws.
Conflict of interest statement
No conflict of interest is declared.
- Areekit S, Khuchareontaworn S, Kanjanavas P, Sriyapai T, Pakpitchareon A, Khawsak P, Chansiri K (2009) Molecular genetics analysis for co-infection of Brugia malayi and Brugia pahangi in cat reservoirs based on internal transcribed spacer region 1. Southeast Asian J Trop Med Public Health 40:30–34PubMedGoogle Scholar
- Chungpivat S, Taweethavonsawat P (2008) The differentiation of microfilariae in dogs and cats using Giemsa’s staining and the detection of acid phosphatase activity. J Thai Vet Pract 20:47–55Google Scholar
- Intapan PM, Thanchomnang T, Lulitanond V, Maleewong W (2009) Rapid detection of Wuchereria bancrofti and Brugia malayi in mosquito vectors (Diptera: Culicidae) using a real-time fluorescence resonance energy transfer multiplex PCR and melting curve analysis. J Med Entomol 46:158–164CrossRefPubMedGoogle Scholar
- World Health Organization (1991) Basic laboratory methods in medical parasitology. World Health Organization, GenevaGoogle Scholar