Simultaneous Use of PCR-RFLP and SSCP Methods for Differentiation of G1 and G3 Genotypes of Echinococcus granulosus sensu stricto

Abstract

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.

Keywords

• Echinococcus granulosus
• genotype
• PCR
• RFLP
• SSCP

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

• Echinococcus granulosus
• genotip
• PZR
• RFLP
• SSCP

Kaynakça

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