Analytical Sensitivity of a Multiplex Quantitative PCR for Toxoplasma Gondii and Neospora Caninum

Overview

Journal Parasitol Res

Specialty Parasitology

Date 2023 Feb 22

PMID 36806974

Authors

Marcus Truong

Jan Slapeta

Affiliations

Soon will be listed here.

Abstract

Cyst-forming coccidia, Toxoplasma gondii and Neospora caninum, are recognised as important causes of animal disease. Molecular diagnostics based on the presence of DNA in animal tissue are required to specifically detect T. gondii and N. caninum while achieving high levels of analytical sensitivity. We optimised available single-plex probe base qPCR assays into a multiplexed qPCR panel to detect cyst-forming coccidia, i.e. T. gondii and N. caninum. The T. gondii assay is based on a 529-bp repetitive (REP) element and the N. caninum assay on the NC5 repetitive region. Using target sequence synthetic DNA, the limit of detection (LOD) was determined to be 100 copies, that is less than a single tachyzoite of either T. gondii or N. caninum. The T. gondii and N. caninum multiplexed qPCR assay optimised in this study can be used to effectively detect parasite DNA for diagnostic purposes in animal tissue.

References

Choi W, Yeom S, Kim J, Jung S, Jung S, Shim T . Hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease. Biosens Bioelectron. 2017; 101:235-244. DOI: 10.1016/j.bios.2017.10.039. View

Galvani A, Christ A, Padula J, Barbosa M, de Araujo R, Sato M . Real-time PCR detection of Toxoplasma gondii in surface water samples in São Paulo, Brazil. Parasitol Res. 2019; 118(2):631-640. DOI: 10.1007/s00436-018-6185-z. View

Barry R, Nissly R, Feria W, Thirumalapura N, Tewari D, Jayarao B . A probe-based real-time PCR assay for the detection of Neospora caninum in clinical samples from cattle. Vet Parasitol. 2019; 269:2-6. DOI: 10.1016/j.vetpar.2019.04.002. View

Steffl M, Nautscher N . Detection of Hammondia heydorni-like oocysts in feces of a dog with recurrent diarrhea. Tierarztl Prax Ausg K Kleintiere Heimtiere. 2019; 47(3):189-192. DOI: 10.1055/a-0890-2350. View

Schares G, Globokar Vrhovec M, Tuschy M, Joeres M, Barwald A, Koudela B . A real-time quantitative polymerase chain reaction for the specific detection of Hammondia hammondi and its differentiation from Toxoplasma gondii. Parasit Vectors. 2021; 14(1):78. PMC: 7830817. DOI: 10.1186/s13071-020-04571-8. View

Pomares C, Estran R, Press C, Bera A, Ramirez R, Montoya J . Is Real-Time PCR Targeting Rep 529 Suitable for Diagnosis of Toxoplasmosis in Patients Infected with Non-Type II Strains in North America?. J Clin Microbiol. 2019; 58(2). PMC: 6989067. DOI: 10.1128/JCM.01223-19. View

Okeoma C, Williamson N, Pomroy W, Stowell K, Gillespie L . The use of PCR to detect Neospora caninum DNA in the blood of naturally infected cows. Vet Parasitol. 2004; 122(4):307-15. DOI: 10.1016/j.vetpar.2004.06.001. View

Belaz S, Gangneux J, Dupretz P, Guiguen C, Robert-Gangneux F . A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii detection by quantitative PCR. J Clin Microbiol. 2015; 53(4):1294-300. PMC: 4365238. DOI: 10.1128/JCM.02900-14. View

Reichel M, Ellis J, Dubey J . Neosporosis and hammondiosis in dogs. J Small Anim Pract. 2007; 48(6):308-12. DOI: 10.1111/j.1748-5827.2006.00236.x. View

10.

Slapeta J, Koudela B, Votypka J, Modry D, Horejs R, Lukes J . Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum. Vet J. 2002; 163(2):147-54. DOI: 10.1053/tvjl.2001.0599. View

11.

Fekkar A, Bodaghi B, Touafek F, Le Hoang P, Mazier D, Paris L . Comparison of immunoblotting, calculation of the Goldmann-Witmer coefficient, and real-time PCR using aqueous humor samples for diagnosis of ocular toxoplasmosis. J Clin Microbiol. 2008; 46(6):1965-7. PMC: 2446865. DOI: 10.1128/JCM.01900-07. View

12.

Allan F, Blake D, Miller Z, Church D . Biliary protozoa in a dog with acute cholangiohepatitis fed a raw food diet. J Vet Intern Med. 2022; 36(6):2177-2180. PMC: 9708427. DOI: 10.1111/jvim.16565. View

13.

Orr B, Ma G, Koh W, Malik R, Norris J, Westman M . Pig-hunting dogs are an at-risk population for canine heartworm (Dirofilaria immitis) infection in eastern Australia. Parasit Vectors. 2020; 13(1):69. PMC: 7020351. DOI: 10.1186/s13071-020-3943-4. View

14.

Muller N, Zimmermann V, Hentrich B, Gottstein B . Diagnosis of Neospora caninum and Toxoplasma gondii infection by PCR and DNA hybridization immunoassay. J Clin Microbiol. 1996; 34(11):2850-2. PMC: 229420. DOI: 10.1128/jcm.34.11.2850-2852.1996. View

15.

Bustin S . Why the need for qPCR publication guidelines?--The case for MIQE. Methods. 2009; 50(4):217-26. DOI: 10.1016/j.ymeth.2009.12.006. View

16.

Ghalmi F, China B, Kaidi R, Daube G, Losson B . Detection of Neospora caninum in dog organs using real time PCR systems. Vet Parasitol. 2008; 155(1-2):161-7. DOI: 10.1016/j.vetpar.2008.04.007. View

17.

Homan W, Vercammen M, De Braekeleer J, Verschueren H . Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int J Parasitol. 2000; 30(1):69-75. DOI: 10.1016/s0020-7519(99)00170-8. View

18.

Peleg O, Baneth G, Eyal O, Inbar J, Harrus S . Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli. Vet Parasitol. 2010; 173(3-4):292-9. DOI: 10.1016/j.vetpar.2010.06.039. View