Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2015 May 21;8(5):366–372. doi: 10.1016/S1995-7645(14)60345-X

Challenging loop—mediated isothermal amplification (LAMP) technique for molecular detection of Toxoplasma gondii

Shirzad Fallahi a,*, Zahra Arab Mazar b, Mehrdad Ghasemian c, Ali Haghighi b
PMCID: PMC7128654  PMID: 26003595

Abstract

Objective

To compare analytical sensitivity and specificity of a newly described DNA amplification technique, LAMP and nested PCR assay targeting the RE and B1 genes for the detection of Toxoplasma gondii (T. gondii) DNA.

Methods

The analytical sensitivity of LAMP and nested-PCR was obtained against10-fold serial dilutions of T. gondii DNA ranging from 1 ng to 0.01 fg. DNA samples of other parasites and human chromosomal DNA were used to determine the specificity of molecular assays.

Results

After testing LAMP and nested-PCR in duplicate, the detection limit of RE-LAMP, B1-LAMP, RE-nested PCR and B1-nested PCR assays was one fg, 100 fg, 1 pg and 10 pg of T. gondii DNA respectively. All the LAMP assays and nested PCRs were 100% specific. The RE-LAMP assay revealed the most sensitivity for the detection of T. gondii DNA.

Conclusions

The obtained results demonstrate that the LAMP technique has a greater sensitivity for detection of T. gondii. Furthermore, these findings indicate that primers based on the RE are more suitable than those based on the B1 gene. However, the B1-LAMP assay has potential as a diagnostic tool for detection of T. gondii.

Keywords: LAMP, Nested-PCR, Molecular detection, Toxoplasma gondii

Footnotes

Available online 20 May 2015

Foundation project: It is supported financially by grant of Lorestan University of Medical Sciences, Khorramabad, Iran.

References

  • 1.Jones JL, Dubey JP. Waterborne toxoplasmosis-recent developments. Exp Parasitol. 2010;124:10–25. doi: 10.1016/j.exppara.2009.03.013. [DOI] [PubMed] [Google Scholar]
  • 2.Kodym P, Malý M, Beran O, Jilich D, Rozsypal H, Machala L. Incidence, immunological and clinical characteristics of reactivation of latent Toxoplasma gondii infection in HIV-infected patients. Epidemiol Infect. 2015;143:600–607. doi: 10.1017/S0950268814001253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Khan A, Su C, German M, Storch GA, Clifford DB, Sibley LD. Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains. J Clin Microbiol. 2005;43:5881–5887. doi: 10.1128/JCM.43.12.5881-5887.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kotresha D, Poonam D, Muhammad Hafiznur Y, Saadatnia G, Nurulhasanah O, Sabariah O. Recombinant proteins from new constructs of SAG1 and GRA7 sequences and their usefulness to detect acute toxoplasmosis. Trop Biomed. 2012;29:129–137. [PubMed] [Google Scholar]
  • 5.Garcia JL, Navarro IT, Vidotto O, Gennari SM, Machado RZ, da Luz Pereira AB. Toxoplasma gondii: comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs. Exp Parasitol. 2006;113:100–105. doi: 10.1016/j.exppara.2005.12.011. [DOI] [PubMed] [Google Scholar]
  • 6.Gangneux FR, Dardé ML. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev. 2012;25:264–296. doi: 10.1128/CMR.05013-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Kodym P, Malý M, Beran O, Jilich D, Rozsypal H, Machala L. Incidence, immunological and clinical characteristics of reactivation of latent Toxoplasma gondii infection in HIV-infected patients. Epidemiol Infect. 2015;143:600–607. doi: 10.1017/S0950268814001253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Olariu TR, Remington JS, Montoya JG. Polymerase chain reaction in cerebrospinal fluid for the diagnosis of congenital toxoplasmosis. Pediatr Infect Dis J. 2014;33:566–570. doi: 10.1097/INF.0000000000000256. [DOI] [PubMed] [Google Scholar]
  • 9.Piergili Fioretti D. Problems and limitations of conventional and innovative methods for the diagnosis of Toxoplasmosis in humans and animals. Parassitologia. 2004;46:177–181. [PubMed] [Google Scholar]
  • 10.Eida OM, Eida MM, Ahmed AB. Evaluation of polymerase chain reaction on amniotic fluid for diagnosis of congenital toxoplasmosis. J Egypt Soc Parasitol. 2009;39:541–550. [PubMed] [Google Scholar]
  • 11.Yu H, Huang B, Zhuo X, Chen X, Du A. Evaluation of a real-time PCR assay based on the single-copy SAG1 gene for the detection of Toxoplasma gondii. Vet Parasitol. 2013;197:670–673. doi: 10.1016/j.vetpar.2013.06.013. [DOI] [PubMed] [Google Scholar]
  • 12.Jalal S, Nord CE, Lappalainen M, Evengård B. Rapid and sensitive diagnosis of Toxoplasma gondii infections by PCR. Clin Microbiol Infect. 2004;10:937–939. doi: 10.1111/j.1469-0691.2004.00948.x. [DOI] [PubMed] [Google Scholar]
  • 13.Kong QM, Lu SH, Tong QB, Lou D, Chen R, Zheng B. Loop-mediated isothermal amplification (LAMP): Early detection of Toxoplasma gondii infection in mice. Parasit Vectors. 2012;3(5):2. doi: 10.1186/1756-3305-5-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Su C, Shwab EK, Zhou P, Zhu XQ, Dubey JP. Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology. 2010;137:1–11. doi: 10.1017/S0031182009991065. [DOI] [PubMed] [Google Scholar]
  • 15.Wiengcharoen JT, Chiabchalard R, Sukthana Y. PCR technique for detecting Toxoplasma gondii in animal amniotic fluid. Southeast Asian J Trop Med Public Health. 2004;2004:792–795. [PubMed] [Google Scholar]
  • 16.Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28:E63. doi: 10.1093/nar/28.12.e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun. 2001;289:150–154. doi: 10.1006/bbrc.2001.5921. [DOI] [PubMed] [Google Scholar]
  • 18.Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cel Prob. 2002;16:223–229. doi: 10.1006/mcpr.2002.0415. [DOI] [PubMed] [Google Scholar]
  • 19.Boehme CC, Nabeta P, Henostroza G, Raqib R, Rahim Z, Gerhardt M. Operational feasibility of using loop-mediated isothermal amplification (LAMP) for the diagnosis of pulmonary TB in microscopy centers of developing countries. J Clin Microbiol. 2007;45:1936–1940. doi: 10.1128/JCM.02352-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem. 2006;52:303–306. doi: 10.1373/clinchem.2005.057901. [DOI] [PubMed] [Google Scholar]
  • 21.Parida M, Posadas G, Inoue S, Hasebe F, Morita K. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J Clin Microbiol. 2004;42:257–263. doi: 10.1128/JCM.42.1.257-263.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Poon LL, Wong BW, Chan KH, Ng SS, Yuen KY, Guan Y. Evaluation of real-time reverse transcriptase PCR and real-time loop-mediated amplification assays for severe acute respiratory syndrome coronavirus detection. J Clin Microbiol. 2005;43:3457–3459. doi: 10.1128/JCM.43.7.3457-3459.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H. Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol. 2003;41:5517–5524. doi: 10.1128/JCM.41.12.5517-5524.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Thekisoe OM, Inoue N, Kuboki N, Tuntasuvan D, Bunnoy W, Borisutsuwan S. Evaluation of loop-mediated isothermal amplification (LAMP), PCR and parasitological tests for detection of Trypanosoma evansi in experimentally infected pigs. Vet Parasitol. 2005;130:327–330. doi: 10.1016/j.vetpar.2005.04.019. [DOI] [PubMed] [Google Scholar]
  • 25.Ikadai H, Tanaka H, Shibahara N, Matsuu A, Uechi M, Itoh N. Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method. J Clin Microbiol. 2004;42:2465–2469. doi: 10.1128/JCM.42.6.2465-2469.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol. 2003;41:2616–2622. doi: 10.1128/JCM.41.6.2616-2622.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Bakheit MA, Torra D, Palomino LA, Thekisoe OM, Mbati PA, Ongerth J. Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing. Vet Parasitol. 2008;158:11–22. doi: 10.1016/j.vetpar.2008.09.012. [DOI] [PubMed] [Google Scholar]
  • 28.Liang SY, Chan YH, Hsia KT, Lee JL, Kuo MC, Hwa KY. Development of loop-mediated isothermal amplification assay for detection of Entamoeba histolytica. J Clin Microbiol. 2009;47:1892–1895. doi: 10.1128/JCM.00105-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Paris DH, Imwong M, Faiz AM, Hasan M, Yunus EB, Silamut K. Loop-mediated isothermal PCR (LAMP) for the diagnosis of falciparum malaria. Am J Trop Med Hyg. 2007;77:972–976. [PubMed] [Google Scholar]
  • 30.Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko H. Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis. J Clin Microbiol. 2007;45:2521–2528. doi: 10.1128/JCM.02117-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Nkouawa A, Sako Y, Nakao M, Nakaya K, Ito A. Loop-mediated isothermal amplification method for differentiation and rapid detection of Taenia species. J Clin Microbiol. 2009;47:168–174. doi: 10.1128/JCM.01573-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Kumagai T, Furushima-Shimogawara R, Ohmae H, Wang TP, Lu S, Chen R. Detection of early and single infections of Schistosoma japonicum in the intermediate host snail, Oncomelania hupensis, by PCR and loop-mediated isothermal amplification (LAMP) assay. Am J Trop Med Hyg. 2010;83:542–548. doi: 10.4269/ajtmh.2010.10-0016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Abbasi I, King CH, Muchiri EM, Hamburger J. Detection of Schistosoma mansoni and Schistosoma haematobium DNA by loop-mediated isothermal amplification: identification of infected snails from early prepatency. Am J Trop Med Hyg. 2010;83:427–432. doi: 10.4269/ajtmh.2010.09-0764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ai L, Li C, Elsheikha HM, Hong SJ, Chen JX, Chen SH. Rapid identification and differentiation of Fasciola hepatica and Fasciola gigantica by a loop-mediated isothermal amplification (LAMP) assay. Vet Parasitol. 2010;174:228–233. doi: 10.1016/j.vetpar.2010.09.005. [DOI] [PubMed] [Google Scholar]
  • 35.Lau YL, Meganathan P, Sonaimuthu P, Thiruvengadam G, Nissapatorn V, Chen Y. Specific, sensitive, and rapid diagnosis of active toxoplasmosis by a loop-mediated isothermal amplification method using blood samples from patients. J Clin Microbiol. 2010;48:3698–3702. doi: 10.1128/JCM.00462-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Krasteva D, Toubiana M, Hartati S, Kusumawati A, Dubremetz JF, Widada JS. Development of loop-mediated isothermal amplification (LAMP) as a diagnostic tool of toxoplasmosis. Vet Parasitol. 2009;162:327–331. doi: 10.1016/j.vetpar.2009.03.014. [DOI] [PubMed] [Google Scholar]
  • 37.Fallahi Sh, Seyyedtabaei SJ, Pournia Y, Zebardast N, Kazemi B. Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia. Diagn Microbiol Infect Dis. 2014;79:347–354. doi: 10.1016/j.diagmicrobio.2014.02.014. [DOI] [PubMed] [Google Scholar]
  • 38.Fallahi Sh, Kazemi B, Seyyedtabaei SJ, Bandehpour M, Lasjerdi Z, Taghipour N. Comparison of the RE and B1 gene for detection of Toxoplasma gondii infection in children with cancer. Parasitol Int. 2014;63:37–41. doi: 10.1016/j.parint.2013.08.005. [DOI] [PubMed] [Google Scholar]
  • 39.Reischl U, Bretagne S, Krüger D, Ernault P, Costa JM. Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect Dis. 2003;2:3–7. doi: 10.1186/1471-2334-3-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Chiabchalard R, Wiengcharoen JT, Sukthana Y. Sensitivity and specificity of PCR for the detection of Toxoplasma gondii and added to laboratory samples. Southeast Asian J Trop Med Pub Health. 2005;36:408–411. [PubMed] [Google Scholar]
  • 41.Zhang H, Thekisoe Oriel MM, Aboge Gabriel O, Kyan H, Yamagishi J, Inoue N. Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method. Exp Parasitol. 2009;122:47–50. doi: 10.1016/j.exppara.2009.01.012. [DOI] [PubMed] [Google Scholar]
  • 42.Lin Z, Zhang Y, Zhang H, Zhou Y, Cao J, Zhou J. Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis. Vet Parasitol. 2012;185:296–300. doi: 10.1016/j.vetpar.2011.10.016. [DOI] [PubMed] [Google Scholar]
  • 43.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) Diag Microbiol Infect Dis. 2008;62:357–365. doi: 10.1016/j.diagmicrobio.2008.07.009. [DOI] [PubMed] [Google Scholar]
  • 44.Costa JM, Pautas C, Ernault P, Foulet F, Cordonnier C, Bretagne S. Real-time PCR for diagnosis and follow-up of Toxoplasma reactivation after allogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes. J Clin Microbiol. 2000;38:2929–2932. doi: 10.1128/jcm.38.8.2929-2932.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Cassaing S, Bessieres MH, Berry A, Berrebi A, Fabre R, Magnaval JF. Comparison between two amplification sets for molecular diagnosis of toxoplasmosis by Real-Time PCR. J Clin Microbiol. 2006;44:720–724. doi: 10.1128/JCM.44.3.720-724.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc. 2008;3:877–882. doi: 10.1038/nprot.2008.57. [DOI] [PubMed] [Google Scholar]
  • 47.Goto M, Honda E, Ogura A, Nomoto A, Hanaki K. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxynaphthol blue. Biotechniques. 2009;46:167–172. doi: 10.2144/000113072. [DOI] [PubMed] [Google Scholar]
  • 48.Mao Z, Qiu Y, Zheng L, Chen J, Yang J. Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea) J Microbiol Methods. 2012;89:179–184. doi: 10.1016/j.mimet.2012.03.011. [DOI] [PubMed] [Google Scholar]

Articles from Asian Pacific Journal of Tropical Medicine are provided here courtesy of Elsevier

RESOURCES