Optimization of Sybr Green Quantitative Real Time Polymerase Chain Reaction (qPCR) using  Excreted-Secreted Antigens (ESAs) Genetik Marker for Detection Toxoplasma gondii

Fitrine Ekawasti; Agus Winarsongko; Farlin Nepho; Eko Setyo Purwanto; Didik Tulus Subekti; harimurti nuradji; NLP Indi Dharmayanti; Riza Zainuddin Ahmad; Siti Sa’diah; Umi Cahyaningsih; Raden Wisnu Nurcahyo

doi:10.22146/jsv.90867

Optimization of Sybr Green Quantitative Real Time Polymerase Chain Reaction (qPCR) using Excreted-Secreted Antigens (ESAs) Genetik Marker for Detection Toxoplasma gondii

https://doi.org/10.22146/jsv.90867

Fitrine Ekawasti^(1*), Agus Winarsongko⁽²⁾, Farlin Nepho⁽³⁾, Eko Setyo Purwanto⁽⁴⁾, Didik Tulus Subekti⁽⁵⁾, harimurti nuradji⁽⁶⁾, NLP Indi Dharmayanti⁽⁷⁾, Riza Zainuddin Ahmad⁽⁸⁾, Siti Sa’diah⁽⁹⁾, Umi Cahyaningsih⁽¹⁰⁾, Raden Wisnu Nurcahyo⁽¹¹⁾

(1) Badan Riset dan Inovasi Nasional
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(*) Corresponding Author

Abstract

Abstract

Toxoplasma gondii is an obligate intracellular parasite, causing toxoplasmosis in almost all warm-blooded animals and humans worldwide. Toxoplasmosis is a zoonotic disease of serious public health concern. Host cell invasion by T. gondii tachyzoites has process involving the sequential secretion of Excreted-Secreted Antigens (ESAs). T. gondi ESAs could be a valuable candidate for the diagnosis of toxoplasmosis. Techniques to more accurately detection of T. gondii recently developed biotechnological methods that are currently being used, conventional and real time Polymerase Chain Reaction (RT-PCR). RT-PCR is more widely used because it is more sensitive and specific. The aims of this study were to optimize the Sybr Green RT-PCR in different region gene based on Excreted-Secreted Antigens (ESAs), tachyzoite surface antigen and bradhyzoite antige, then adapt the conventional PCR program to real-time PCR for detection Toxoplasma gondii. Optimization is necessary to get optimal condition of PCR to get the best results. T. gondii RH strains derived from liquid nitrogen and DNA extracted by DNAzol. The genetic marker used GRA1#1, GRA1#2, GRA7#1, GRA7#2, ROP1, MIC3, SAG1 and BAG1. The results of the optimization of multiple primer genes can adapt and be used optimal in RT-PCR by using the same cycle program simultaneously in one run. Overall, RT-PCR for the detection of T. gondii DNA demonstrated excellent agreement with conventional PCR. RT-PCR with melting curve analysis is rapid and simple that facilitates high throughput analysis to detect T. gondii. The optimal conditions obtained from the optimization results can facilitate further research to detect T. gondii.

Keywords: Biotechnology molecular, Detection, excretory-secretory antigen, toxoplasmosis

Keywords

Biotechnology molecular; Detection; excretory-secretory antigen; toxoplasmosis

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References

References Azimpour-Ardakan T, Fotouhi-Ardakani R, Hoghooghi-Rad N, Rokni N, Motallebi A. (2021). Phylogenetic Analysis and Genetics Polymorphisms Evaluation of ROP8 and B1 Genes of Toxoplasma gondii in Livestock and Poultry Hosts of Yazd, Qom and Golestan Provinces of Iran. Iranian journal of parasitology. 16 4 pp.576–586. Apsari, I., Artama, W.S. and Damriyasa, I. (2012) Molecular diagnosis of Toxoplasma gondii based on the tachyzoite and bradyzoite stage specific genes in free-range chicken. J. Vet., 13(1): 14-19. Beraki T., Hu X., Broncel M., Young J.C., O’Shaughnessy W.J., Borek D., Treeck M., Reese M.L. (2019). Divergent kinase regulates membrane ultrastructure of the Toxoplasma parasitophorous vacuole. Proc. Natl. Acad. Sci. USA. 2019; 116:6361–6370. Braun, L., Brenier-Pinchart, M. P., Yogavel, M., Curt-Varesano, A., Curt-Bertini, R. L., Hussain, T., Kieffer-Jaquinod, S., Coute, Y., Pelloux, H., Tardieux, I., Sharma, A., Belrhali, H., Bougdour, A., & Hakimi, M. A. (2013). A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation. The Journal of experimental medicine, 210(10), 2071–2086. https://doi.org/10.1084/jem.20130103 Brenier-Pinchart, M.P., Morand-Bui, V., Fricker-Hidalgo, H., Equy, V., Marlu, R., Pelloux, H., 2007. Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control. Parasite Paris Fr. 14, 149–154 Cai D, Behrmann O, Hufert F, Dame G, Urban G. (2018). Capacity of rTth polymerase to detect RNA in the presence of various inhibitors. PLoS ONE 13(1): e0190041. https://doi.org/10.1371/journal.pone.0190041. Calderaro A, Piccolo G, Gorrini C, Peruzzi S, Zerbini L, Bommezzadri S, Dettori G, Chezzi C. (2006). Comparison between two Real- time PCR assays and a nested PCR for the detection of Toxoplasma gondii. Acta Bio Med. 2006; 77(2):75-80. Chatzidimopoulos M, Ganopoulos I, Madesis P, Vellios E, Tsaftaris A, Pappas A C. (2014). High-resolution melting analysis for rapid detection and characterization ofBotrytis cinereaphenotypes resistant to fenhexamid and boscalid. Plant Pathology 63(6): 1336-1343. Costa JM, Cabaret O, Moukoury S, Bretagne S. (2011). Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene. J Microbiol Methods. 2011; 86:357–63 Dai F, Zhao X, Tang C, Wang Z, Kuang Z, Li Z, Huang J and Luo G (2018) Identification and validation of reference genes for qRT‐PCR analysis in mulberry (Morus alba L.). PLoS One 13, e0194129. Ekawasti F, Cahyaningsih U, Dharmayanti NLPI, Sa'diah S, Subekti DT, Azmi Z, Desem MI (2021) Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI, Int. J. One Health, 7(2):196-203.. Ekawasti, F., PurwantoE. S., NephoF., KurniawatiD. A., SubektiD. T., DamayantiR., Sa’diahS., & CahyaningsihU. (2023). The Patterns of Restriction Fragment of Several Enzymes to Distinguish Toxoplasma gondii Isolates Virulent and Avirulent Strains using GRA1 and GRA7 Genetic Marker. HAYATI Journal of Biosciences, 30(4), 725-733. https://doi.org/10.4308/hjb.30.4.725-733 El Aal AAA, Nahnoush RK, Elmallawany MA, El-Sherbiny WS, Badr MS, Nasr GM (2018) Isothermal PCR for feasible molecular diagnosis of primary toxoplasmosis in women recently experienced spontaneous abortion. Open Access Maced J Med Sci 6(6):982–987 Fatimi, 2010. Quantitative real time PCR for the measurement of WSSV in shrimp. J. Fish Dis. 25, 381-389. Fehlberg S, Allen J, Church K. (2013). A novel method of genomic DNA extraction for Cactaceae Applications in plant sciences. 1 10.3732/apps.1200013. Franco M, Panas M W, Marino N D, Lee M C, Buchholz K R, Kelly F D, Bednarski J J, Sleckman, B P, Pourmand N, Boothroyd. (2018). A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells. Mbio. 2018 Oct;9(5): e01869-18. DOI: 10.1128/mbio.01869-18. PMID: 30279290; PMCID: PMC6168865. Gallup, J. M., & Ackermann, M. R. (2006). Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently highfidelity qPCR reactions. Biological procedures online, 8, 87–152. https://doi.org/10.1251/bpo122 Ghafari S M, Fotouhi-Ardakani R, Parvizi P. (2020). Designing and developing a high-resolution melting technique for accurate identification of Leishmania species by targeting amino acid permease 3 and cytochrome oxidase II genes using real-time PCR and in silico genetic evaluation. Acta Trop. 211 p. 105626. Gold D A, Kaplan A D, Lis A, Bett G C, Rosowski E E, Cirelli K M, Bougdour A, Sidik S M, Beck J R, Lourido S, Egea P F, Bradley P J, Hakimi M A, Rasmusson R L, Saeij J P. (2015). The Toxoplasma Dense Granule Proteins GRA17 and GRA23 Mediate the Movement of Small Molecules between the Host and the Parasitophorous Vacuole. Cell Host & Microbe. 2015 May;17(5):642-652. DOI: 10.1016/j.chom.2015.04.003. PMID: 25974303; PMCID: PMC4435723 Gudnason, H., Dufva, M., Bang, D. D., & Wolff, A. (2007). Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic acids research, 35(19), e127. https://doi.org/10.1093/nar/gkm671 Hosseini-Safa, A., Shojaee, S., Salami, S. A., Mohebali, M., Hantoushzadeh, S., Mousavi, P., Dehghan Manshadi, S. A., & Keshavarz Valian, H. (2020). Development of High Resolution Melting Analysis as a Diagnostic Tool for Molecular Detection of Toxoplasma Infection in Pregnant Women and HIV Positive Cases. Iranian journal of public health, 49(10), 1983–1991. https://doi.org/10.18502/ijph.v49i10.4704 Hou S, Liu Y, Tang Y, Wu M, Guan J, Li X, Wang Z, Jiang J, Deng M, Duan Z, Tang X, Han X, Jiang L. (2019). Anti-Toxoplasma gondii effect of two spider venoms in vitro and in vivo. Toxicon. 2019; 166:9–14. doi: 10.1016/j.toxicon.2019.05.003 Indrasanti, D. Haryanto, A., Artama, W. (2011). Expression and Isolation of Recombinant Microneme 3 (MIC3) Protein of Toxoplasma gondii Local Isolate on Eschericia coli (BL21). Animal Production. 13 2 pp.131–137. Ivovic, V., Marija Vujanic, M., Tijana, Š., Ivana, K. and Olgica, D.D. (2012) Molecular detection and genotyping of Toxoplasma gondii from clinical samples. Rec. Adv., 10(5772): 50830. Kralik, P., & Ricchi, M. (2017). A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Frontiers in microbiology, 8, 108. https://doi.org/10.3389/fmicb.2017.00108. Kuang, J., Yan, X., Genders, A. J., Granata, C., & Bishop, D. J. (2018). An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research. PloS one, 13(5), e0196438. https://doi.org/10.1371/journal.pone.0196438 Kusumaningsih, P. (2018) Evaluasi Konstruksi DNA Da+Lam Vektor Plasmid Berkaitan Dengan Ekspresi Protein Rekombinan Rophtry-1 (ROP1) Toxoplasma Gondii Pada Eschericia coli. Seminar Ilmiah Nasional Teknologi, Sains, dan Sosial Humaniora, Bali. Lieveld M, Carregosa A, Benoy I, Redzic N, Berth M, Broeck DV. (2017). A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV2. J. Virol. Methods. 248 pp.181–186. Marino, A. M., Percipalle, M., Giunta, R. P., Salvaggio, A., Caracappa, G., Alfonzetti, T., Aparo, A., & Reale, S. (2017). Development and validation of a real-time PCR assay for the detection of Toxoplasma gondii DNA in animal and meat samples. Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 29(2), 203–207. https://doi.org/10.1177/1040638716682808 Moncada, P. A., & Montoya, J. G. (2012). Toxoplasmosis in the fetus and newborn: an update on prevalence, diagnosis and treatment. Expert review of anti-infective therapy, 10(7), 815–828. https://doi.org/10.1586/eri.12.58 Nagy, N. A., Németh, Z., Juhász, E., Póliska, S., Rácz, R., Kosztolányi, A., & Barta, Z. (2017). Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae). PeerJ, 5, e4047. https://doi.org/10.7717/peerj.4047 Nolan T, Huggett J, Sanchez E. Good practice guide for the application of quantitative PCR (qPCR). LGC. 2013. Natl Meas Syst. 50 Nuryady M M, Husamah H, Miharja F J, Hindun I, Patmawati P 2020 J. Penelitian Dan Pengkajian Ilmu Pendidikan: E-Saintika. 4 2 pp. 223–233. Opsteegh, M., Kortbeek, T. M., Havelaar, A. H., & van der Giessen, J. W. (2015). Intervention strategies to reduce human Toxoplasma gondii disease burden. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 60(1), 101–107. https://doi.org/10.1093/cid/ciu721 Papini R., Di Ciccio P., Marangi M., Ghidini S., Zanardi E., Vergara A., Giangaspero A., Nardoni S., Rocchigiani G., Mancianti F., (2017). Occurrence of Toxoplasma gondii in Carcasses of Pigs Reared in Intensive Systems in Northern Italy. J. Food Prot. 2017; 80:515–522. doi: 10.4315/0362-028X.JFP-16-314. Puspita OS, Yasmon A, Dewi BE. (2020). Optimizing Real-Time PCR methods for detection of ssaN gene Salmonella enterica subsp.enterica in the blood specimen. Journal of Biomedicine and Translational Research. 2020 Aug;6(2):41-47. https://doi.org/10.14710/jbtr.v6i2.7120. Ramamurthy, M., Alexander, M., Aaron, S., Kannangai, R., Ravi, V., Sridharan, G., & Abraham, A. M. (2011). Comparison of a conventional polymerase chain reaction with real-time polymerase chain reaction for the detection of neurotropic viruses in cerebrospinal fluid samples. Indian journal of medical microbiology, 29(2), 102–109. https://doi.org/10.4103/0255-0857.81777 Romand, S., Chosson, M., Franck, J., Wallon, M., Kieffer, F., Kaiser, K., Dumon, H., Peyron, F., Thulliez, P., & Picot, S. (2004). Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii. American journal of obstetrics and gynecology, 190(3), 797–802 Rommereim LM, Fox BA, Butler KL, Cantillana V, Taylor GA, Bzik DJ. (2019). Rhoptry and Dense Granule Secreted Effectors Regulate CD8+ T Cell Recognition of Toxoplasma gondii Infected Host Cells. Front Immunol. 2019 Sep 6; 10:2104. doi: 10.3389/fimmu.2019.02104. PMID: 31555296; PMCID: PMC6742963. Samarut E, Bekri A and Drapeau P. (2016). Transcriptomic Analysis of Purified Embryonic Neural Stem Cells from Zebrafish Embryos Reveals Signaling Pathways Involved in Glycine-Dependent Neurogenesis. Front. Mol. Neurosci. 9:22. doi: 10.3389/fnmol.2016.00022 Schares, G., Globokar Vrhovec, M., Tuschy, M., Joeres, M., Bärwald, A., Koudela, B., Dubey, J. P., Maksimov, P., & Conraths, F. J. (2021). A real-time quantitative polymerase chain reaction for the specific detection of Hammondia hammondi and its differentiation from Toxoplasma gondii. Parasites & vectors, 14(1), 78. https://doi.org/10.1186/s13071-020-04571-8 Schrader, C., Schielke, A., Ellerbroek, L., & Johne, R. (2012). PCR inhibitors - occurrence, properties and removal. Journal of applied microbiology, 113(5), 1014–1026. https://doi.org/10.1111/j.1365-2672.2012.05384.x Słomka, M., Sobalska-Kwapis, M., Wachulec, M., Bartosz, G., & Strapagiel, D. (2017). High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies. International journal of molecular sciences, 18(11), 2316. https://doi.org/10.3390/ijms18112316 Subekti DT, WT Artama, E Sulistyaningsih, SH Poerwanto, Y Sari dan F Bagaskoro. 2008. Kloning dan analisis hasil kloning gen GRA1 dari Takizoit Toxoplasma gondii isolat lokal. JITV 13(1), 44–52. Switaj K, Master A, Skrzypczak M, Zaborowski P. (2005). Recent trends in molecular diagnostics for Toxoplasma gondii infections. Clin. Microbiol. Infect. 11 pp.170–176. von Ahlfen, S. and Schlumpberger, M., (2010) Qiagen Gene Expression Newsletter, 15: 7–8. Wang, Z. D., Wang, S. C., Liu, H. H., Ma, H. Y., Li, Z. Y., Wei, F., Zhu, X. Q., & Liu, Q. (2017). Prevalence and burden of Toxoplasma gondii infection in HIV-infected people: a systematic review and meta-analysis. The lancet. HIV, 4(4), e177–e188. https://doi.org/10.1016/S2352-3018(17)30005-X Wei, W., Zhang, F., Chen, H. (2018). Toxoplasma gondii dense granule protein 15 induces apoptosis in choriocarcinoma JEG-3 cells through endoplasmic reticulum stress. Parasites Vectors 11, 251 (2018). https://doi.org/10.1186/s13071-018-2835-3 Yu H, Huang B, Zhuo X, et al. Evaluation of a real-time PCR assay based on the single-copy SAG1 gene for the detection of Toxoplasma gondii. Vet Parasitol. 2013;197(3):670–673. doi: 10.1016/j.vetpar.2013.06.013 Zhao, F., Maren, N. A., Kosentka, P. Z., Liao, Y. Y., Lu, H., Duduit, J. R., Huang, D., Ashrafi, H., Zhao, T., Huerta, A. I., Ranney, T. G., & Liu, W. (2021). An optimized protocol for stepwise optimization of real-time RT-PCR analysis. Horticulture research, 8(1), 179. https://doi.org/10.1038/s41438-021-00616-w Zhu, W., Li, J., Pappoe, F., Shen, J., & Yu, L. (2019). Strategies Developed by Toxoplasma gondii to Survive in the Host. Frontiers in microbiology, 10, 899. https://doi.org/10.3389/fmicb.2019.00899

DOI: https://doi.org/10.22146/jsv.90867