Research Article
Development of toxoplasma gondii 529 base pair size repeat region (RE) specific rapid loop mediated isothermal amplification test and determination of analytical sensitivity
Abstract
Aim: Toxoplasma gondii is a protozoon that causes disease in humans and warm-blooded animals. The aim of this study is to determine the analytical sensitivity of the fluorescence-based rapid loop mediated isothermal amplification (LAMP) test developed by designing primers specific to T. gondii repeat region (RE) for the diagnosis of toxoplasmosis.
Materials and Methods: T. gondii RE obtained with PCR was cloned into vector pCR2.1-TOPO. The analytical sensitivity of the LAMP assay developed using specific primers was determined by serial dilutions of the pCR2.1-RE vector
Results: It was determined that the analytical sensitivity of the LAMP test developed with primers designed specific to T. gondii RE gene was 10 copy plasmid / reaction.
Conclusion: Since the RE region repeats 200-300 times in the T. gondii gene, the test developed has been calculated to have a detection limit of 0.05 tachyzoites/reaction. The LAMP test is predicted to be promising because it is easy, cheap, fast and suitable for field conditions and is more sensitive than other molecular tests
References
- Dubey J., Miller N., J.K F. The Toxoplasma gondii oocyst from cat feces. J Exp Med. 1970; 132 (4): 636–62.
- Karakavuk M, Aldemir D, Mercier A, Şahar EA, Can H, Murat JB, et al. Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey. PLoS One. 2018; 13 (4): 1–17.
- Rajendran C, Su C, Dubey JP. Molecular genotyping of Toxoplasma gondii from Central and South America revealed high diversity within and between populations. Infect Genet Evol. 2012; 12 (2): 359–68.
- Velmurugan G V., Dubey JP, Su C. Genotyping studies of Toxoplasma gondii isolates from Africa revealed that the archetypal clonal lineages predominate as in North America and Europe. Vet Parasitol. 2008; 155 (3–4): 314–8.
- Harma M, Harma M, Güngen N, Demir N. Toxoplasmosis in pregnant women in Şanlıurfa, Southeastern Anatolia City, Turkey. J Egypt Soc Parasitol. 2004; 34: 519–25.
- Holland GN. Ocular toxoplasmosis: A global reassessment. Part I: Epidemiology and course of disease. Am J Ophthalmol. 2003; 136 (6): 973–88.
- Elbez-Rubinstein A, Ajzenberg D, Dardé ML, Cohen R, Dumètre A, Yera H, et al. Congenital toxoplasmosis and reinfection during pregnancy: case report, strain characterization, experimental model of reinfection, and review. J Infect Dis. 2009; 199 (2): 280–5.
- Fallahi S, Kazemi B, Seyyed tabaei SJ, Bandehpour M, Lasjerdi Z, Taghipour N, et al. Comparison of the RE and B1 gene for detection of Toxoplasma gondii infection in children with cancer. Parasitol Int [Internet]. 2014; 63 (1): 37–41. Available from: http://dx.doi.org/10.1016/j.parint.2013.08.005
- Yuan Z, Gao S, Liu Q, Xia X, Liu X, Liu B, et al. Toxoplasma gondii antibodies in cancer patients. Cancer Lett. 2007; 254 (1): 71–4.
- Remington JS, Thulliez P, Montoya JG. Recent Developments for Diagnosis of Toxoplasmosis. J Clin Microbiol. 2004; 42 (3): 941–5.
- Kolören Z, Avşar C, Şekeroǧlu ZA. [Diagnosis of protozoa by loop-mediated isothermal amplification: (LAMP)]. Turkiye Parazitol Derg. 2010; 34 (4): 207–11.
- Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res [Internet]. 2000; 28 (12): e63.
- Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002; 16 (3): 223–9.
- Edvinsson B, Lappalainen M, Evengård B, Buffalano W, Ferguson D, Guy E, et al. Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis. Clin Microbiol Infect. 2006; 12 (2): 131–6.
- Homan W., Vercammen M, Braekeleer J De, 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.
- Sağlam Metiner P, Can H, Ayyıldız Tamiş D, Karakavuk M, Kımız Geboloğlu I, Gülçe İz S, et al. The use of Toxoplasma gondii tachyzoites produced in HeLa cells adhered to Cytodex 1 microcarriers as antigen in serological assays: an application of microcarrier technology. Cytotechnology. 2019; 71 (1): 91–105.
- Can H, Inceboz T, Caner A, Atalay Şahar E, Karakavuk M, Döşkaya M, et al. Kist Örneklerinde Yeni Bir Tek Tüp Multipleks Gerçek Zamanli Polimeraz Zincir Reaksiyonu He Echinococcus granulosus ve Echinococcus multilocularis’ in Saptanmasi. Mikrobiyol Bul. 2016; 50 (2): 266–77.
- Döşkaya M, Caner A, Deǧirmenci A, Wengenack NL, Yolasiǧmaz A, Turgay N, et al. Degree and frequency of inhibition in a routine realtime PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey. J Med Microbiol. 2011; 60 (7): 937–44.
- Li Y, Fan P, Zhou S, Zhang L. Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens. Microb Pathog [Internet]. 2017; 107: 54–61. Available from: http://dx.doi.org/10.1016/j.micpath.2017.03.016
- Zhang H, Thekisoe OMM, Aboge GO, Kyan H, Yamagishi J, Inoue N, et al. Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method. Exp Parasitol [Internet]. 2009;122(1):47–50. Available from: http://dx.doi.org/10.1016/j.exppara.2009.01.012
- 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 [Internet]. 2008; 62:357–65. Available from: http://dx.doi.org/10.1016/j.diagmicrobio.2008.07.009
- Yang Q, Zhang R, Wu H, Zhang Y, Wang K. Detection of Toxoplasma gondii DNA by loop-mediated isothermal amplification. Chinese J Parasitol Parasit Dis. 2008; 26 (4): 304–6.
- Fallahi S, Mazar ZA, Ghasemian M, Haghighi A. Challenging loop-mediated isothermal amplification (LAMP) technique for molecular detection of Toxoplasma gondii. Asian Pac J Trop Med [Internet]. 2015;8(5):366–72. Available from: http://dx.doi.org/10.1016/S1995-7645 (14) 60345-X
- Iwasaki M, Yonekawa T, Otsuka K, Suzuki W, Nagamine K, Hase T, et al. Validation of the loop-mediated isothermal amplification method for single nucleotide polymorphism genotyping with whole blood. Genome Lett. 2003; (2): 119–26
Toxoplasma gondii 529 baz çifti büyüklüğünde tekrar bölgesine (RE) özgü hızlı döngü aracılı izotermal amplifikasyon testinin geliştirilmesi ve analitik hassasiyetinin belirlenmesi
Abstract
Amaç: Toxoplasma gondii insan ve sıcakkanlı hayvanlarda hastalıklara neden olan bir protozoondur. Bu çalışmanın amacı toksoplazmozis tanısı için T. gondii tekrar bölgesine (RE) özgü primerlerin tasarlanması ile geliştirilen floresans esaslı hızlı Döngü Aracılı İzotermal Amplifikasyon (LAMP) testinin analitik hassasiyetini belirlemektir.
Gereç ve Yöntem: Polimeraz Zincir Reaksiyonu (PZR) ile elde edilen T. gondii RE, pCR2.1-TOPO vektörüne klonlanmıştır. Spesifik primerler kullanılarak geliştirilen LAMP testinin analitik hassasiyeti pCR2.1-RE vektörünün seri seyreltmeleri ile belirlenmiştir.
Bulgular: T. gondii RE genine özgü tasarlanan primerler ile geliştirilen LAMP testinin analitik hassasiyetinin ≤10 plazmit/reaksiyon olduğu tespit edilmiştir.
Sonuç: RE bölgesi T. gondii geni içinde 200-300 kere tekrar ettiğinden dolayı geliştirilen testin 0,05 takizoit/reaksiyon saptama limitine sahip olduğu hesaplanmıştır. LAMP testinin kolay, ucuz, hızlı ve saha koşullarına uygun olması ve diğer moleküler testlerden daha hassas olması nedeniyle umut verici olduğu öngörülmektedir
Keywords
References
- Dubey J., Miller N., J.K F. The Toxoplasma gondii oocyst from cat feces. J Exp Med. 1970; 132 (4): 636–62.
- Karakavuk M, Aldemir D, Mercier A, Şahar EA, Can H, Murat JB, et al. Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey. PLoS One. 2018; 13 (4): 1–17.
- Rajendran C, Su C, Dubey JP. Molecular genotyping of Toxoplasma gondii from Central and South America revealed high diversity within and between populations. Infect Genet Evol. 2012; 12 (2): 359–68.
- Velmurugan G V., Dubey JP, Su C. Genotyping studies of Toxoplasma gondii isolates from Africa revealed that the archetypal clonal lineages predominate as in North America and Europe. Vet Parasitol. 2008; 155 (3–4): 314–8.
- Harma M, Harma M, Güngen N, Demir N. Toxoplasmosis in pregnant women in Şanlıurfa, Southeastern Anatolia City, Turkey. J Egypt Soc Parasitol. 2004; 34: 519–25.
- Holland GN. Ocular toxoplasmosis: A global reassessment. Part I: Epidemiology and course of disease. Am J Ophthalmol. 2003; 136 (6): 973–88.
- Elbez-Rubinstein A, Ajzenberg D, Dardé ML, Cohen R, Dumètre A, Yera H, et al. Congenital toxoplasmosis and reinfection during pregnancy: case report, strain characterization, experimental model of reinfection, and review. J Infect Dis. 2009; 199 (2): 280–5.
- Fallahi S, Kazemi B, Seyyed tabaei SJ, Bandehpour M, Lasjerdi Z, Taghipour N, et al. Comparison of the RE and B1 gene for detection of Toxoplasma gondii infection in children with cancer. Parasitol Int [Internet]. 2014; 63 (1): 37–41. Available from: http://dx.doi.org/10.1016/j.parint.2013.08.005
- Yuan Z, Gao S, Liu Q, Xia X, Liu X, Liu B, et al. Toxoplasma gondii antibodies in cancer patients. Cancer Lett. 2007; 254 (1): 71–4.
- Remington JS, Thulliez P, Montoya JG. Recent Developments for Diagnosis of Toxoplasmosis. J Clin Microbiol. 2004; 42 (3): 941–5.
- Kolören Z, Avşar C, Şekeroǧlu ZA. [Diagnosis of protozoa by loop-mediated isothermal amplification: (LAMP)]. Turkiye Parazitol Derg. 2010; 34 (4): 207–11.
- Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res [Internet]. 2000; 28 (12): e63.
- Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002; 16 (3): 223–9.
- Edvinsson B, Lappalainen M, Evengård B, Buffalano W, Ferguson D, Guy E, et al. Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis. Clin Microbiol Infect. 2006; 12 (2): 131–6.
- Homan W., Vercammen M, Braekeleer J De, 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.
- Sağlam Metiner P, Can H, Ayyıldız Tamiş D, Karakavuk M, Kımız Geboloğlu I, Gülçe İz S, et al. The use of Toxoplasma gondii tachyzoites produced in HeLa cells adhered to Cytodex 1 microcarriers as antigen in serological assays: an application of microcarrier technology. Cytotechnology. 2019; 71 (1): 91–105.
- Can H, Inceboz T, Caner A, Atalay Şahar E, Karakavuk M, Döşkaya M, et al. Kist Örneklerinde Yeni Bir Tek Tüp Multipleks Gerçek Zamanli Polimeraz Zincir Reaksiyonu He Echinococcus granulosus ve Echinococcus multilocularis’ in Saptanmasi. Mikrobiyol Bul. 2016; 50 (2): 266–77.
- Döşkaya M, Caner A, Deǧirmenci A, Wengenack NL, Yolasiǧmaz A, Turgay N, et al. Degree and frequency of inhibition in a routine realtime PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey. J Med Microbiol. 2011; 60 (7): 937–44.
- Li Y, Fan P, Zhou S, Zhang L. Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens. Microb Pathog [Internet]. 2017; 107: 54–61. Available from: http://dx.doi.org/10.1016/j.micpath.2017.03.016
- Zhang H, Thekisoe OMM, Aboge GO, Kyan H, Yamagishi J, Inoue N, et al. Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method. Exp Parasitol [Internet]. 2009;122(1):47–50. Available from: http://dx.doi.org/10.1016/j.exppara.2009.01.012
- 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 [Internet]. 2008; 62:357–65. Available from: http://dx.doi.org/10.1016/j.diagmicrobio.2008.07.009
- Yang Q, Zhang R, Wu H, Zhang Y, Wang K. Detection of Toxoplasma gondii DNA by loop-mediated isothermal amplification. Chinese J Parasitol Parasit Dis. 2008; 26 (4): 304–6.
- Fallahi S, Mazar ZA, Ghasemian M, Haghighi A. Challenging loop-mediated isothermal amplification (LAMP) technique for molecular detection of Toxoplasma gondii. Asian Pac J Trop Med [Internet]. 2015;8(5):366–72. Available from: http://dx.doi.org/10.1016/S1995-7645 (14) 60345-X
- Iwasaki M, Yonekawa T, Otsuka K, Suzuki W, Nagamine K, Hase T, et al. Validation of the loop-mediated isothermal amplification method for single nucleotide polymorphism genotyping with whole blood. Genome Lett. 2003; (2): 119–26
There are 24 citations in total.
Details
| Primary Language | Turkish |
|---|---|
| Subjects | Health Care Administration |
| Journal Section | Research Articles |
| Authors | |
| Publication Date | September 13, 2021 |
| Submission Date | January 13, 2021 |
| Published in Issue | Year 2021Volume: 60 Issue: 3 |
