Staphylococcus aureus izolatlarının tiplendirilmesinde çoklu lokus değişken sayı tekrar analizi (MLVA) ve darbeli alan jel elektroforez (PFGE) yöntemlerinin karşılaştırılması

Yıl 2020, Cilt: 22 Sayı: 2, 679 - 686, 10.04.2020

Sümeyra Savaş

https://doi.org/10.25092/baunfbed.755053

Öz

Bu çalışmada, Staphylococcus aureus (S. aureus) bakterisi için, çoklu lokus değişken sayı tekrar analizi (MLVA) yönteminin, altın standart olarak bilinen moleküler tiplendirme yöntemi darbeli alan jel elektroforezi (PFGE) ile ayırt edebilme performansı araştırıldı. S. aureus için PFGE yönteminden elde edilen sonuçların MLVA yöntemi ile ne oranda sağlanabileceği belirlenmeye çalışıldı. Bu sayede rutin kullanımlarda zorlu bir yöntem olan PFGE yönteminin, daha kolay ve düşük maliyetli bir yöntem olan MLVA ile rutin de yer değiştirmesinin olanağı araştırıldı.
MLVA yöntemi hızlı bir DNA izolasyon yöntemi sonrası, sdrCDE, clfA, clfB, sspA ve spa lokusları için polimeraz zincir reaksiyonu (PCR) yöntemi kullanılarak gerçekleştirildi. PFGE yöntemi ise SmaI enzimi kullanılarak elde edilen genomik DNA fragmanları ile gerçekleştirildi. Bantlama modeli, hem görsel olarak hem de Bionumerics yazılımı ile analiz edildi. PFGE ile izolatlar 2 küme ve 16 alt tipe ayrılırken, MLVA ile aynı izolatlar, küme oluşturmaksızın 31 alt tipe ayrıldı. PCR temelli bu yöntem ile, PFGE yöntemi ile ayırt edilemeyen alt tiplerin ayırt edilebildiği görüldü. Bu sebep ile MLVA yönteminin aynı PFGE paternleri ile ilgisiz suşları daha iyi tanımlamak veya ilişkili izolatların yakın genetik kompozisyonunu doğrulamak için yüksek epidemiyolojik çözünürlüğe sahip, alternatif, verimli moleküler tipleme yöntemi olarak kullanılabileceği sonucuna varıldı.

Anahtar Kelimeler

Çoklu lokus değişken sayı tekrar analizi (MLVA), darbeli alan jel elektroforezi (PFGE), ayırım gücü, moleküler tiplendirme, Staphylococcus aureus

Teşekkür

Çalışma da kullanılan örneklerin teminindeki desteklerinden ötürü Doç. Dr. Gülşen Hazırolan’a ve analizlerde ki desteğinden ötürü Doç. Dr. Alper Karagöz’e teşekkürlerimi sunarım.

Comparison of multiple locus variable number of tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis methods for typing Staphylococcus aureus isolates

Öz

In this study, the performance of multi locus variable number tandem repeat analysis (MLVA) method to distinguish pulsed-field gel electrophoresis (PFGE) types, known as the gold standard, for Staphylococcus aureus (S. aureus). It was tried to determine to what extent the results obtained from PFGE method for S. aureus can be obtained with MLVA method. In this way, the possibility of displacement of PFGE method, which is a challenging method in routine use, with MLVA, which is an easier and low cost method has been investigated.
MLVA method was performed using a polymerase chain reaction (PCR) method for sdrCDE, clfA, clfB, sspA and spa after a rapid DNA isolation method. The PFGE method was performed with genomic DNA fragments obtained using the SmaI enzyme. The banding model was analyzed both visually and with the Bionumerics software. While isolates were divided into 2 clusters and 16 subtypes with PFGE, the same izolates were divided into 31 subtypes without forming a cluster with MLVA method. With this PCR- based method, it was seen that the strains indistinguishable by PFGE method could be distinguished. Therefore, it was concluded thet MLVA can be used as an alternative, efficient molecular typing method with high epidemiological resolution to beter identify strains unrelated to the same PFGE patterns or to verify the close genetic composition of related isolates.

Anahtar Kelimeler

Multiple-locus variable-number tandem-repeat analysis (MLVA), Pulsed-field gel electrophoresis (PFGE), discrimination power, molecular typing, Staphylococcus aureus

Kaynakça

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