Advertisement

Parasitology Research

, Volume 116, Issue 4, pp 1151–1157 | Cite as

Development of T m -shift genotyping method for detection of cat-derived Giardia lamblia

  • Weida Pan
  • Yeqi Fu
  • Auwalu Yusuf Abdullahi
  • Mingwei Wang
  • Xianli Shi
  • Fang Yang
  • Xingang Yu
  • Xinxin Yan
  • Pan Zhang
  • Jianxiong Hang
  • Guoqing LiEmail author
Original Paper

Abstract

To develop T m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5′-end were designed according to two SNPs (BG434 and BG170) of β-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52 × 10−5 and 4.88 × 10−5 ng/μL samples of assemblage A and F standard plasmids. The T m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.

Keywords

Giardia lamblia Cat Tm-shift genotyping SNP β-giardin gene 

Notes

Acknowledgements

This work was supported by grant from National Natural Science Foundation of China (grant nos. 31272551, 31672541). The authors would like to thank Prof. Zhaorong Lun from Southern China Research Center of Parasitic Biology, Sun Yat-sen University, China for offering G. lamblia trophozoites assemblages A.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Ahn JJ, Kim Y, Hong JY, Kim GW, Kim SY, Hwang SY (2016) Probe-based fluorescence melting curve analysis for differentiating Larimichthys polyactis and Larimichthys crocea. Food Anal Method 9(7):2036–2041CrossRefGoogle Scholar
  2. Caccio SM, Ryan U (2008) Molecular epidemiology of giardiasis. Mol Biochem Parasitol 160(2):75–80CrossRefPubMedGoogle Scholar
  3. Cacciò SM, Thompson RCA, Mclauchlin J, Smith HV (2005) Unravelling Cryptosporidium and Giardia epidemiology. Trends Parasitol 21(9):430–437CrossRefPubMedGoogle Scholar
  4. Cardozo GP, Santos EV, Fachin AL, Franca SC, Marins M (2011) A glass bead protocol for recovery of host cell free Ehrlichia canis and quantification by Sybr-green real-time PCR. Biocell 35(1):35–36PubMedGoogle Scholar
  5. Chen H, Parimelalagan M, Lai YL, Lee KS, Koay ES, Hapuarachchi HC, Ng LC, Ho PS, Chu JH (2015) Development and evaluation of a SYBR Green-based real-time multiplex RT-PCR assay for simultaneous detection and serotyping of dengue and chikungunya viruses. J Mol Diagn 17(6):722–728CrossRefPubMedGoogle Scholar
  6. Choy SH, Al-Mekhlafi HM, Mahdy MAK, Nasr NN, Sulaiman M, Lim YAL, Surin J (2014) Prevalence and associated risk factors of Giardia infection among indigenous communities in rural Malaysia. Sci Rep-UK 4(6909)Google Scholar
  7. Dery V, Duah NO, Ayanful-Torgby R, Matrevi SA, Anto F, Quashie NB (2015) An improved SYBR Green-I-based fluorescence method for the routine monitoring of Plasmodium falciparum resistance to anti-malarial drugs. Malaria J 14(481)Google Scholar
  8. Derzelle S, Mendy C, Laroche S, Madani N (2011) Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination. J Microbiol Meth 87(2):195–201CrossRefGoogle Scholar
  9. Feng Y, Xiao L (2011) Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 24(1):110–140CrossRefPubMedPubMedCentralGoogle Scholar
  10. Huang Y, Nie S, Zhou S, Li K, Sun J, Zhao J, Fei B, Wang Z, Ye H, Hong Q, Gao X, Duan S (2015) Polymorphism and circulating lipid levels connect with brain diseases in Han Chinese and suggest sex-dependent effects. Biomed Pharmacother 70:7–11CrossRefPubMedGoogle Scholar
  11. Hunter PR, Thompson R (2005) The zoonotic transmission of Giardia and Cryptosporidium. Int J Parasitol 35(11–12):1181–1190CrossRefPubMedGoogle Scholar
  12. Karanis P, Ey PL (1998) Characterization of axenic isolates of Giardia intestinasis established from humans and animals in Germany. Parasitol Res 84:442–449CrossRefPubMedGoogle Scholar
  13. Kumar JS, Saxena D, Panda M (2014) Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients. Mol Cell Probe 28(5–6):221–227CrossRefGoogle Scholar
  14. Lalle M, Pozio E, Capelli G, Bruschi F, Crotti D, Caccio SM (2005) Genetic heterogeneity at the beta-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. Int J Parasitol 35(2):207–213CrossRefPubMedGoogle Scholar
  15. Lasek-Nesselquist E, Welch DM, Sogin ML (2010) The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems. Int J Parasitol 40(9):1063–1074CrossRefPubMedPubMedCentralGoogle Scholar
  16. Lebbad M, Mattsson JG, Christensson B, Ljungstrom B, Backhans A, Andersson JO, Svard SG (2010) From mouse to moose: multi locus genotyping of Giardia isolates from various animal species. Vet Parasitol 168(3–4):231–239CrossRefPubMedGoogle Scholar
  17. Lefebvre SL, Waltner-Toews D, Peregrine AS, Reid-Smith R, Hodge L, Arroyo LG, Weese JS (2006) Prevalence of zoonotic agents in dogs visiting hospitalized people in Ontario: implications for infection control. J Hosp Inf 62(4):458–466CrossRefGoogle Scholar
  18. Li J, Zhang P, Wang P, Alsarakibi M, Zhu H, Liu Y, Meng X, Li J, Guo J, Li G (2012) Genotype identification and prevalence of Giardia duodenalis in pet dogs of Guangzhou, Southern China. Vet Parasitol 188(3–4):368–371CrossRefPubMedGoogle Scholar
  19. Navarro L, Gongora C, Benavides P (2010) Single nucleotide polymorphism detection at the Hypothenemus hampei Rdl gene by allele-specific PCR amplification with T m-shift primers. Pestic Biochem Phys 97(3):204–208CrossRefGoogle Scholar
  20. Ponchel F, Toomes C, Bransfield K, Leong FT, Douglas SH, Field SL, Bell SM, Combaret V, Puisieux A, Mighell AJ, Robinson PA, Inglehearn CF, Isaacs JD, Markham AF (2003) Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol 3(18)Google Scholar
  21. Qi M, Yu F, Li S, Wang H, Luo N, Huang J, Zhang L (2015) Multilocus genotyping of potentially zoonotic Giardia duodenalis in pet chinchillas (Chinchilla lanigera) in China. Vet Parasitol 208(3–4):113–117CrossRefPubMedGoogle Scholar
  22. Ryan U, Caccio SM (2013) Zoonotic potential of Giardia. Int J Parasitol 43(12–13):943–956CrossRefPubMedGoogle Scholar
  23. Shastry BS (2007) SNPs in disease gene mapping, medicinal drug development and evolution. J Hum Genet 52(11):871–880CrossRefPubMedGoogle Scholar
  24. Sotiriadou I, Pantchev N, Gassmann D, Karanis P (2013) Molecular identification of Giardia and Cryptosporidium from dogs and cats. Parasite 20:8CrossRefPubMedPubMedCentralGoogle Scholar
  25. Suzuki J, Murata R, Kobayashi S, Sadamasu K, Kai A, Takeuchi T (2011) Risk of human infection with Giardia duodenalis from cats in Japan and genotyping of the isolates to assess the route of infection in cats. Parasitology 138(4):493–500CrossRefPubMedGoogle Scholar
  26. Tan L, Yu X, Abdullahi AY, Wu S, Zheng G, Hu W, Song M, Wang Z, Jiang B, Li G (2015) Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia. Parasitol Res 114(11):4081–4086CrossRefPubMedGoogle Scholar
  27. Tan L, Wu S, Abdullahi AY, Yu X, Hu W, Song M, Shi X, Li G (2016) PCR-RFLP method to detect zoonotic and host-specific Giardia duodenalis assemblages in dog fecal samples. Parasitol Res 115(5):2045–2050CrossRefPubMedGoogle Scholar
  28. Thompson RC, Hopkins RM, Homan WL (2000) Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol Today 16:210–213CrossRefPubMedGoogle Scholar
  29. Trout JM, Santin M, Fayer R (2007) Prevalence of Giardia duodenalis genotypes in adult dairy cows. Vet Parasitol 147:205–209CrossRefPubMedGoogle Scholar
  30. Wang J, Chuang K, Ahluwalia M, Patel S, Umblas N, Mirel D, Higuchi R, Germer S (2005) High-throughput SNP genotyping by single-tube PCR with T m-shift primers. BioTechniques 39(6):885–893CrossRefPubMedGoogle Scholar
  31. Zhang P, Liu Y, Alsarakibi M, Li J, Liu T, Li Y, Li G (2012) Application of HRM assays with EvaGreen dye for genotyping Giardia duodenalis zoonotic assemblages. Parasitol Res 111(5):2157–2163CrossRefPubMedGoogle Scholar
  32. Zheng G, Hu W, Liu Y, Luo Q, Tan L, Li G (2015) Occurrence and molecular identification of Giardia duodenalis from stray cats in Guangzhou, Southern China. Korean J Parasitol 53(1):119–124CrossRefPubMedPubMedCentralGoogle Scholar
  33. Zhou S, Liu M, An W, Liang X, Yu W, Gong B, Piao F (2012a) Genotyping of human platelet antigen-15 by single closed-tube T m-shift method. Int J Lab Hematol 34(1):41–46CrossRefPubMedGoogle Scholar
  34. Zhou S, Liu M, An W, Liang X, Yu W, Gong B, Piao F (2012b) Genotyping of human platelet antigen-15 by single closed-tube T m-shift method. Int J Lab Hematol 34(1):41–46CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Weida Pan
    • 1
  • Yeqi Fu
    • 1
  • Auwalu Yusuf Abdullahi
    • 1
  • Mingwei Wang
    • 1
  • Xianli Shi
    • 1
  • Fang Yang
    • 1
  • Xingang Yu
    • 1
  • Xinxin Yan
    • 1
  • Pan Zhang
    • 1
  • Jianxiong Hang
    • 1
  • Guoqing Li
    • 1
    Email author
  1. 1.Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary MedicineSouth China Agricultural UniversityGuangzhouChina

Personalised recommendations