DOI QR코드

DOI QR Code

Sensitive and Rapid Detection of Giardia lamblia Infection in Pet Dogs using Loop-Mediated Isothermal Amplification

  • Li, Jie (College of Veterinary Medicine, South China Agricultural University) ;
  • Wang, Peiyuan (College of Veterinary Medicine, South China Agricultural University) ;
  • Zhang, Aiguo (College of Veterinary Medicine, South China Agricultural University) ;
  • Zhang, Ping (College of Veterinary Medicine, South China Agricultural University) ;
  • Alsarakibi, Muhamd (College of Veterinary Medicine, South China Agricultural University) ;
  • Li, Guoqing (College of Veterinary Medicine, South China Agricultural University)
  • 투고 : 2012.09.27
  • 심사 : 2013.01.10
  • 발행 : 2013.04.30

초록

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from $10^{-1}$ to $10^{-5}ng/{\mu}l$ for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of $63^{\circ}C$ by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha ($EF1{\alpha}$) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

키워드

참고문헌

  1. Appelbee AJ, Thompson RC, Olson ME. Giardia and Cryptosporidium in mammalian wildlife-current status and future needs. Trends Parasitol 2005; 21: 370-376. https://doi.org/10.1016/j.pt.2005.06.004
  2. Becher KA, Robertson ID, Fraser DM, Palmer DG, Thompson RC. Molecular epidemiology of Giardia and Cryptosporidium infections in dairy calves originating from three sources in Western Australia. Vet Parasitol 2004; 123: 1-9. https://doi.org/10.1016/j.vetpar.2004.05.020
  3. Ballweber LR, Xiao L, Bowman DD, Kahn G, Cama VA. Giardiasis in dogs and cats: update on epidemiology and public health significance. Trends Parasitol 2010; 26: 180-189. https://doi.org/10.1016/j.pt.2010.02.005
  4. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011; 24: 110-140. https://doi.org/10.1128/CMR.00033-10
  5. Thompson RC. The zoonotic significance and molecular epidemiology of Giardia and giardiasis. Vet Parasitol 2004; 126: 15-35. https://doi.org/10.1016/j.vetpar.2004.09.008
  6. Thompson J, Yang R, Power M, Hufschmid J, Beveridge I, Reid S, Ng J, Armson A, Ryan U. Identification of zoonotic Giardia genotypes in marsupials in Australia. Exp Parasitol 2008; 120: 88-93. https://doi.org/10.1016/j.exppara.2008.05.002
  7. Caccio SM, Thompson RC, McLauchlin J, Smith HV. Unravelling Cryptosporidium and Giardia epidemiology. Trends Parasitol 2005; 21: 430-437. https://doi.org/10.1016/j.pt.2005.06.013
  8. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000; 28: e63. https://doi.org/10.1093/nar/28.12.e63
  9. Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 2001; 289: 150-154. https://doi.org/10.1006/bbrc.2001.5921
  10. Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, Sugimoto C, Igarashi I. Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol 2003; 41: 5517-5524. https://doi.org/10.1128/JCM.41.12.5517-5524.2003
  11. Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, Peiris JS. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heattreated blood by loop-mediated isothermal amplification. Clin Chem 2006; 52: 303-306.
  12. Karanis P, Thekisoe O, Kiouptsi K, Ongerth J, Igarashi I, Inoue N. Development and preliminary evaluation of a loop-mediated isothermal amplification procedure for sensitive detection of cryptosporidium oocysts in fecal and water samples. Appl Environ Microbiol 2007; 73: 5660-5662. https://doi.org/10.1128/AEM.01152-07
  13. Sotiriadou I, Karanis P. Evaluation of loop-mediated isothermal amplification for detection of Toxoplasma gondii in water samples and comparative findings by polymerase chain reaction and immunofluorescence test (IFT). Diagn Microbiol Infect Dis 2008; 62: 357-365. https://doi.org/10.1016/j.diagmicrobio.2008.07.009
  14. Plutzer J, Karanis P. Rapid identification of Giardia duodenalis by loop-mediated isothermal amplification (LAMP) from faecal and environmental samples and comparative findings by PCR and real-time PCR methods. Parasitol Res 2009; 104: 1527-1533. https://doi.org/10.1007/s00436-009-1391-3
  15. Plutzer J, Torokne A, Karanis P. Combination of ARAD microfibre filtration and LAMP methodology for simple, rapid and cost-effective detection of human pathogenic Giardia duodenalis and Cryptosporidium spp. in drinking water. Lett Appl Microbiol 2010; 50: 82-88. https://doi.org/10.1111/j.1472-765X.2009.02758.x
  16. Li J, Wang PY, Zhang P, Liu YJ, Guo JC, Meng XL, Li GQ. Cloning and sequence analysis of $EF1{\alpha}$ gene of the Giardia lamblia from dogs. Prog Vet Med 2012; 33: 53-56 (in Chinese).
  17. Nagamine K, Hase T, Notomi T. Accelerated reaction by loopmediated isothermal amplification using loop primers. Mol Cell Probes 2002; 16: 223-229. https://doi.org/10.1006/mcpr.2002.0415

피인용 문헌

  1. Diagnostic testing for Giardia infections vol.108, pp.3, 2013, https://doi.org/10.1093/trstmh/tru005
  2. Loop-mediated isothermal amplification assay for detection of Histomonas meleagridis infection in chickens targeting the 18S rRNA sequences vol.43, pp.1, 2013, https://doi.org/10.1080/03079457.2013.873112
  3. Molecular Detection of Giardia intestinalis from Stray Dogs in Animal Shelters of Gyeongsangbuk-do (Province) and Daejeon, Korea vol.53, pp.4, 2015, https://doi.org/10.3347/kjp.2015.53.4.477
  4. Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Fecal Samples vol.2016, pp.None, 2013, https://doi.org/10.1155/2016/7376207
  5. Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis vol.10, pp.1, 2017, https://doi.org/10.1186/s13071-017-2330-2
  6. Sensitive and rapid detection of Ortleppascaris sinensis (Nematoda: Ascaridoidea) by loop-mediated isothermal amplification vol.7, pp.None, 2013, https://doi.org/10.7717/peerj.7607