Echinococcus granulosus sensu stricto’nun G1 ve G3 Genotiplerinin Ayrımında PZR-RFLP ve SSCP Metotlarının Eş Zamanlı Kullanımı
Öz
Kistik ekinokokkozis (KE), dünya çapında insan ve hayvanlarda yaygın olarak rastlanan ihmal edilmiş bir halk sağlığı ve ekonomik sorundur. Bu çalışmanın amacı, E. granulosus s.s. (G1/G3) izolatları içerisinde G1 ve G3 genotiplerinin PZR-Restriction Fragment Length Polymorphism (PZR-RFLP) ve takiben Single Stranded Conformation Polymorphism (SSCP) teknikleriyle ayrımının yapılmasıdır. Çalışmada kullanılan insan, sığır ve koyun hidatik kist izolatlarına ait genomik DNA (gDNA)’lar E. granulosus s.s. (G1/G3) olduğu DNA dizi analizi ile teyit edilen örnekler arasından seçilmiştir. Genomik DNA’lar, E. granulosus s.s.’nun G1 ve G3 genotiplerini ayırt eden mt-nad5 bölgesini çoğaltan primerler kullanılarak PZR ile çoğaltıldı. Bu PZR ürünleri MboII, HindII ve HphI restriksiyon enzimleriyle ayrı ayrı kesildi. Daha sonra hazırlanan SSCP jeline bu kesim ürünleri yüklendi ve elektroforeze tabi tutulup jel gümüş nitrat ile boyanarak bantlar görünür hale getirildi. Bu çalışmada hepsi Elazığ ilinden elde edilen ikisi insan, altısı sığır ve 13’ü koyun olmak üzere toplam 21 izolat analiz edildi. Bütün örneklerde E. granulosus s.s.’un 759 bp’lik mt-nad5 gen bölgesi PZR ile başarılı bir şekilde çoğaltıldı. RFLP neticesinde MboII enzimi G1 ve G3 genotiplerine ait PZR ürünlerini dört farklı pozisyondan, HindII iki farklı ve yine HphI de iki farklı pozisyondan kesti. Takiben yapılan SSCP analizinde bütün örneklerin aynı bant profilini göstermesi nedeniyle E. granulosus s.s.’nun G1 ve G3 genotiplerinin bu yöntemle ayırt edilemeyeceği anlaşıldı.
Anahtar Kelimeler
Kaynakça
- Rickard MD, Lightowlers MW (1986). The Biology of Echinococcus and Hydatid Disease (In): Immunodiagnosis of Hydatid Disease. Thompson RCA (Editors). George Allen and Unwin. London, England., 217-249.
- Kesik HK, Simsek S, Kilinc SG, Koroglu E (2019). Identification of Antigen B (AgB) gene polymorphism in cattle and sheep isolates of Echinococcus granulosus and investigation of effects on serological diagnosis. Acta Trop., 199:105099.
- Simsek S, Balkaya I, Koroglu E (2010). Epidemiological survey and molecular characterization of Echinococcus granulosus in cattle in an endemic area of eastern Turkey. Vet Parasitol., 172(3-4):347-349.
- Utuk AE, Simsek S, Koroglu E, McManus DP (2008). Molecular genetic characterization of different isolates of Echinococcus granulosus in east and southeast regions of Turkey. Acta Trop., 107(2):192-194.
- Kinkar L, Laurimae T, Acosta-Jamett G, et al. (2018). Distinguishing Echinococcus granulosus sensu stricto genotypes G1 and G3 with confidence: a practical guide. Infect Genet Evol., 64:178-184.
- Yanagida T, Mohammadzadeh T, Kamhawi S, et al. (2012). Genetic polymorphisms of Echinococcus granulosus sensu stricto in the Middle East. Parasitol Int., 61(4):599–603.
- Kinkar L, Laurimae T, Simsek S, et al. (2016). High-resolution phylogeography of zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1 with an emphasis on its distribution in Turkey, Italy and Spain. Parasitol., 143(13):1790-1801.
- Laurimae T, Kinkar L, Andresiuk V, et al. (2016). Genetic diversity and phylogeography of highly zoonotic Echinococcus granulosus genotype G1 in the Americas (Argentina, Brazil, Chile and Mexico) based on 8279 bp of mtDNA. Infect Genet Evol., 45:290-296.
- Simsek S, Roinioti E, Eroksuz H (2015). First report of Echinococcus equinus in a donkey in Turkey. Korean J Parasitol., 53(6): 731-735.
- Avcioglu H, Guven E, Balkaya I, et al. (2021). The situation of echinococcosis in stray dogs in Turkey: the first finding of Echinococcus multilocularis and Echinococcus ortleppi. Parasitol., 148(9):1092-1098.
- Cengiz G, Gonenc B (2020). Comparison of molecular and morphological characterization and haplotype analysis of cattle and sheep isolates of cystic echinococcosis. Vet Parasitol., 282: 109132.
- Celik F, Selcuk MA, Kilinc SG, et al. (2024). Molecular discrimination of G1 and G3 genotypes of Echinococcus granulosus sensu stricto obtained from human, cattle, and sheep using the mitochondrial NADH dehydrogenase subunit 5 marker. Acta Trop., 252:107124.
- Simsek S, Balkaya I, Ciftci AT, Utuk AE (2011). Molecular discrimination of sheep and cattle isolates of Echinococcus granulosus by SSCP and conventional PZR in Turkey. Vet Parasitol., 178(3-4):367-369.
- Gulija TK, Ivancic-Jelecki J, Santak M, Forcic D (2011). Comparative analysis of CE-SSCP to standard RFLP-CE-FLA method in quantification of known viral variants within an RNA virus quasispecies. Electrophoresis., 32(14):1852-1859.
- Jabbar A, Gasser RB (2013). Mutation scanning analysis of genetic variation within and among Echinococcus species: implications and future prospects. Electrophoresis., 34(13):1852-1862.
- Gasser RB, Hu M, Chilton NB, et al. (2006). Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation. Nat Protoc., 1(6):3121-3128.
- Gasser RB (2006). Molecular tools-advances, opportunities and prospects. Vet Parasitol., 136(2):69-89.
- Gasser RB (2013). A perfect time to harness advanced molecular technologies to explore the fundamental biology of Toxocara species. Vet Parasitol., 193(4):353-364.
- Hashim HO, Al-Shuhaib MBS (2019). Exploring the potential and limitations of PZR-RFLP and PZR-SSCP for SNP detection: A review. J Appl Biotechnol Rep., 6(4):137-144.
- Botstein D, White RL, Skolnick M, Davis RW (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet., 32(2):314-331.
- Zhang L, Gasser RB, Zhu X, McManus DP (1999). Screening for different genotypes of Echinococcus granulosus within China and Argentina by single-strand conformation polymorphism (SSCP) analysis. Trans R Soc Trop Med Hyg., 93(3):329-334.
- Oudni-M’rad M, Cabaret J, M’rad S, et al. (2006). Genetic differences between Tunisian camel and sheep strains of the cestode Echinococcus granulosus revealed by SSCP. Parasite., 13(2):131-136.
Simultaneous Use of PCR-RFLP and SSCP Methods for Differentiation of G1 and G3 Genotypes of Echinococcus granulosus sensu stricto
Öz
Cystic echinococcosis (CE) is a neglected public health and economic problem affecting both humans and animals worldwide. The study aimed to differentiate G1 and G3 genotypes among E. granulosus s.s. (G1/G3) isolates using PCR-Restriction fragment length polymorphism (PCR-RFLP) followed by Single Stranded Conformation Polymorphism (SSCP) techniques. The study selected genomic DNA (gDNA) from confirmed E. granulosus s.s. (G1/G3) human, cattle, and sheep hydatid cyst isolates. PCR was used to amplify the mt-nad5 region of E. granulosus s.s. with primers that distinguish G1 and G3 genotypes. The resulting PCR products were digested separately with MboII, HindII, and HphI restriction enzymes. The digestion products were then loaded onto the prepared SSCP gel and subjected to electrophoresis. Finally, the gel was stained with silver nitrate to make the bands visible. The study analysed a total of 21 isolates, comprising two human, six cattle, and 13 sheep, all obtained from Elazig province of Türkiye. The PCR successfully amplified the 759 bp mt-nad5 gene region of E. granulosus s.s. in all samples. As a result of RFLP, MboII enzyme cut the PCR products of G1 and G3 genotypes at four different positions, HindII at two different positions and HphI at two different positions. The subsequent SSCP analysis revealed that the G1 and G3 genotypes of E. granulosus s.s. could not be differentiated using this method, as all samples displayed an identical band profile.
Anahtar Kelimeler
Kaynakça
- Rickard MD, Lightowlers MW (1986). The Biology of Echinococcus and Hydatid Disease (In): Immunodiagnosis of Hydatid Disease. Thompson RCA (Editors). George Allen and Unwin. London, England., 217-249.
- Kesik HK, Simsek S, Kilinc SG, Koroglu E (2019). Identification of Antigen B (AgB) gene polymorphism in cattle and sheep isolates of Echinococcus granulosus and investigation of effects on serological diagnosis. Acta Trop., 199:105099.
- Simsek S, Balkaya I, Koroglu E (2010). Epidemiological survey and molecular characterization of Echinococcus granulosus in cattle in an endemic area of eastern Turkey. Vet Parasitol., 172(3-4):347-349.
- Utuk AE, Simsek S, Koroglu E, McManus DP (2008). Molecular genetic characterization of different isolates of Echinococcus granulosus in east and southeast regions of Turkey. Acta Trop., 107(2):192-194.
- Kinkar L, Laurimae T, Acosta-Jamett G, et al. (2018). Distinguishing Echinococcus granulosus sensu stricto genotypes G1 and G3 with confidence: a practical guide. Infect Genet Evol., 64:178-184.
- Yanagida T, Mohammadzadeh T, Kamhawi S, et al. (2012). Genetic polymorphisms of Echinococcus granulosus sensu stricto in the Middle East. Parasitol Int., 61(4):599–603.
- Kinkar L, Laurimae T, Simsek S, et al. (2016). High-resolution phylogeography of zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1 with an emphasis on its distribution in Turkey, Italy and Spain. Parasitol., 143(13):1790-1801.
- Laurimae T, Kinkar L, Andresiuk V, et al. (2016). Genetic diversity and phylogeography of highly zoonotic Echinococcus granulosus genotype G1 in the Americas (Argentina, Brazil, Chile and Mexico) based on 8279 bp of mtDNA. Infect Genet Evol., 45:290-296.
- Simsek S, Roinioti E, Eroksuz H (2015). First report of Echinococcus equinus in a donkey in Turkey. Korean J Parasitol., 53(6): 731-735.
- Avcioglu H, Guven E, Balkaya I, et al. (2021). The situation of echinococcosis in stray dogs in Turkey: the first finding of Echinococcus multilocularis and Echinococcus ortleppi. Parasitol., 148(9):1092-1098.
- Cengiz G, Gonenc B (2020). Comparison of molecular and morphological characterization and haplotype analysis of cattle and sheep isolates of cystic echinococcosis. Vet Parasitol., 282: 109132.
- Celik F, Selcuk MA, Kilinc SG, et al. (2024). Molecular discrimination of G1 and G3 genotypes of Echinococcus granulosus sensu stricto obtained from human, cattle, and sheep using the mitochondrial NADH dehydrogenase subunit 5 marker. Acta Trop., 252:107124.
- Simsek S, Balkaya I, Ciftci AT, Utuk AE (2011). Molecular discrimination of sheep and cattle isolates of Echinococcus granulosus by SSCP and conventional PZR in Turkey. Vet Parasitol., 178(3-4):367-369.
- Gulija TK, Ivancic-Jelecki J, Santak M, Forcic D (2011). Comparative analysis of CE-SSCP to standard RFLP-CE-FLA method in quantification of known viral variants within an RNA virus quasispecies. Electrophoresis., 32(14):1852-1859.
- Jabbar A, Gasser RB (2013). Mutation scanning analysis of genetic variation within and among Echinococcus species: implications and future prospects. Electrophoresis., 34(13):1852-1862.
- Gasser RB, Hu M, Chilton NB, et al. (2006). Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation. Nat Protoc., 1(6):3121-3128.
- Gasser RB (2006). Molecular tools-advances, opportunities and prospects. Vet Parasitol., 136(2):69-89.
- Gasser RB (2013). A perfect time to harness advanced molecular technologies to explore the fundamental biology of Toxocara species. Vet Parasitol., 193(4):353-364.
- Hashim HO, Al-Shuhaib MBS (2019). Exploring the potential and limitations of PZR-RFLP and PZR-SSCP for SNP detection: A review. J Appl Biotechnol Rep., 6(4):137-144.
- Botstein D, White RL, Skolnick M, Davis RW (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet., 32(2):314-331.
- Zhang L, Gasser RB, Zhu X, McManus DP (1999). Screening for different genotypes of Echinococcus granulosus within China and Argentina by single-strand conformation polymorphism (SSCP) analysis. Trans R Soc Trop Med Hyg., 93(3):329-334.
- Oudni-M’rad M, Cabaret J, M’rad S, et al. (2006). Genetic differences between Tunisian camel and sheep strains of the cestode Echinococcus granulosus revealed by SSCP. Parasite., 13(2):131-136.
Ayrıntılar
| Birincil Dil | Türkçe |
|---|---|
| Konular | Veteriner Parazitoloji |
| Bölüm | Araştıma |
| Yazarlar | |
| Yayımlanma Tarihi | 30 Haziran 2025 |
| Gönderilme Tarihi | 12 Şubat 2025 |
| Kabul Tarihi | 14 Mayıs 2025 |
| Yayımlandığı Sayı | Yıl 2025 Cilt: 18 Sayı: 1 |
