Parasitology Research

, Volume 106, Issue 3, pp 621–625 | Cite as

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

  • Tongjit Thanchomnang
  • Pewpan M. IntapanEmail author
  • Sudchit Chungpivat
  • Viraphong Lulitanond
  • Wanchai Maleewong
Original Paper


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.


Fluorescence Resonance Energy Transfer Melting Curve Analysis Babesia Differential Detection Restriction Fragment Length Polymorphism Profile 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



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.


  1. 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
  2. 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
  3. Dissanaike AS (1979) Zoonotic aspects of filarial infections in man. Bull World Health Organ 57:349–357PubMedGoogle Scholar
  4. 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
  5. Lulitanond V, Intapan PM, Pipitgool V, Choochote W, Maleewong W (2004) Rapid detection of Wuchereria bancrofti in mosquitoes by LightCycler polymerase chain reaction and melting curve analysis. Parasitol Res 94:337–341CrossRefPubMedGoogle Scholar
  6. Lyon E, Wittwer CT (2009) LightCycler technology in molecular diagnostics. J Mol Diagn 11:93–101CrossRefPubMedGoogle Scholar
  7. Mak JW, Yen PK, Lim KC, Ramiah N (1980) Zoonotic implications of cats and dogs in filarial transmission in Peninsular Malaysia. Trop Geogr Med 32:259–264PubMedGoogle Scholar
  8. McReynolds LA, DeSimone SM, Williams SA (1986) Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi. Proc Natl Acad Sci USA 83:797–801CrossRefPubMedGoogle Scholar
  9. Nuchprayoon S, Junpee A, Poovorawan Y, Scott AL (2005) Detection and differentiation of filarial parasites by universal primers and polymerase chain reaction–restriction fragment length polymorphism analysis. Am J Trop Med Hyg 73:895–900PubMedGoogle Scholar
  10. Palmieri JR, Ratiwayanto S, Masbar S, Tirtokusumo S, Rusch J, Marwoto HA (1985) Evidence of possible natural infections of man with Brugia pahangi in South Kalimantan (Borneo), Indonesia. Trop Geogr Med 37:239–244PubMedGoogle Scholar
  11. Rao RU, Weil GJ, Fischer K, Supali T, Fischer P (2006) Detection of Brugia parasite DNA in human blood by real-time PCR. J Clin Microbiol 44:3887–3893CrossRefPubMedGoogle Scholar
  12. Sivanandam S, Fredericks HJ (1966) The “Innenkorper” in differentiation between the microfilariae of Brugia pahangi and B. malayi (sub-periodic form). Med J Malaya 20:337–338PubMedGoogle Scholar
  13. Sucharit S, Harinasuta C, Viraboonchai S, Smithanonda S (1975) The differentiation of Brugia malayi, B. pahangi, B. tupaiae and Wuchereria bancrofti. Southeast Asian J Trop Med Public Health 6:549–554PubMedGoogle Scholar
  14. Thanchomnang T, Intapan PM, Lulitanond V, Choochote W, Manjai A, Prasongdee TK, Maleewong W (2008) Rapid detection of Brugia malayi in mosquito vectors using a real-time fluorescence resonance energy transfer PCR and melting curve analysis. Am J Trop Med Hyg 78:509–513PubMedGoogle Scholar
  15. World Health Organization (1991) Basic laboratory methods in medical parasitology. World Health Organization, GenevaGoogle Scholar
  16. Yen PK, Mak JW (1978) Histochemical differentiation of Brugia, Wuchereria, Dirofilaria and Breinlia microfilariae. Ann Trop Med Parasitol 72:157–162PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Tongjit Thanchomnang
    • 1
  • Pewpan M. Intapan
    • 1
    Email author
  • Sudchit Chungpivat
    • 3
  • Viraphong Lulitanond
    • 2
  • Wanchai Maleewong
    • 1
  1. 1.Department of Parasitology, Faculty of MedicineKhon Kaen UniversityKhon KaenThailand
  2. 2.Department of Microbiology, Faculty of Medicine and Research and Diagnostic Center for Emerging Infectious DiseasesKhon Kaen UniversityKhon KaenThailand
  3. 3.Parasitology Unit, Department of Pathology, Faculty of Veterinary ScienceChulalongkorn UniversityBangkokThailand

Personalised recommendations