MURAT ŞEVİK, OĞUZHAN AVCI, ÖMER BARIŞ İNCE

Comparison of manual and automated nucleic acid extraction methods for detection of peste des petits ruminants virus RNA

Koyun ve keçi vebası (PPR), küçük ruminantların ekonomik açıdan önemli, bulaşıcı bir hastalığıdır. Günümüzde PCR tabanlı teknikler, PPRın hızlı tanısı için başarıyla kullanılmaktadır. PPR virusunun (PPRV) tespitinde reverse transkripsiyon PCR (RT-PCR) metotları kullanılır iken, doku örneklerinden RNA izolasyonu için kullanılan yöntem önem arz etmektedir. Bu çalışmada, RNA ekstraksiyonu için ticari manuel bir kit ve otomatik bir işleme tekniği performans açısından karşılaştırılmıştır. Laboratuvara PPR şüphesi ile gönderilen herbiri farklı sürüden otuz iki küçükbaş hayvan, PPRV tespitinde manuel ve otomatik ekstraksiyon yöntemlerinin karşılaştırılması için seçilmiştir. Vero hücreleri PPRV izolasyonu için kullanılmıştır. PPRV RNAsının tespiti için one step RT-PCR metodu kullanılmıştır. Otuz iki örnekten, 11 âdetinde sitopatojenik efekt (CPE) gözlenmiştir. Manuel ekstraksiyon metodu ile 32 örneğin 11inde PPRV nükleik asidi tespit edilirken, robot kullanılarak yapılan otomatik ekstraksiyon metodu ile 9 örnekte viral RNA tespit edilmiştir. Otomatik ekstraksiyon metodu ile negatif tespit edilen 2 örnek, manuel ekstraksiyon metodu sonucu zayıf pozitif olarak tespit edilmiştir. Manuel ve otomatik ekstraksiyon sonucu elde edilen RNA miktarı ve kalitesi spektrofotometre cihazı kullanılarak karşılaştırılmıştır. Elde edilen sonuçlara göre, iki metot arasında elde edilen RNA miktar farkı istatiksel olarak önemli bulunmuş (P

Peste des petits ruminants virus RNAsının tespitinde manuel ve otomatik nükleik asit ekstraksiyon yöntemlerinin karşılaştırılması

Peste des petits ruminants (PPR) is an economically important contagious disease of small ruminants. PCR-based techniques have been successfully used for rapid diagnosis of PPR. The method used for isolation of RNA from tissue samples is an important concern when using reverse transcription-PCR (RT-PCR) methods for the detection of PPR virus (PPRV). In this study, a commercial kit for manual preparation and an automated processing technique for RNA extraction were compared in terms of performance. Thirty-two small ruminants, each from different flocks, with PPR suspect submitted to laboratory were chosen to compare manual and automated extraction methods for the detection of PPRV. Vero cells were used for PPRV isolation. One-step RT-PCR was used for the detection of PPRV RNA. From the 32 submitted samples, CPE was observed in 11 samples. PPRV nucleic acid was detected in 11 of 32 samples that were manually extracted, while viral RNA was detected in 9 of 32 extracts prepared by the robot. Two samples that were negative with automated extraction were weakly positive in manual extraction. RNA quality and quantity were assessed using a spectrophotometer. According to the results, difference in quantity among two methods was statistically significant (P<0.0001, two-tailed paired t-test), and manual extraction method is suitable for detection of low amounts of PPRV RNA in clinical samples.

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