Development of real-time reverse transcriptase-polymerase chain reaction (rt RT- PCR) targeting four genes of peste des petits ruminants virus

Abstract

In this study, one-step real-time quantitative reverse transcription PCR (qRT- PCR) was developed for the first time and evaluated for detection of peste des petits ruminants virus (PPRV) in field samples obtained from distinct geographical areas of Turkey. Primers and probes targeting four PPRV genes (namely L, M, N and P) were designed based on full genome sequence (AJ849636) of the local isolate (Tu00). The detection limits of the assays were found to be 7 copies/µL RNA for L, 6 copies/µL RNA for P, 10 copies/µL RNA for M and 700 copies/µL RNA for N, respectively. Besides, the detection ratio for PPRV in 45 field samples was 100% (45) for L, 88.9% (40) for M, 77.8% (35) for P and 22.3% (10) for N, respectively. In conclution, onestep real-time quantitative reverse transcription PCR (qRT- PCR) assay reported here for L gene design provides rapid, specific and sensitive detection of PPRV in tissue samples obtained from field cases

Keywords

• Peste des petits ruminants
• Real-time qRT-PCR
• virus detection

Küçük ruminant vebasının dört genini hedefleyen gerçek zamanlı reverz - transkriptaz polimerazzincir reaksiyonu (rtRT – PZR)’ nun geliştirilmesi

Abstract

Bu çalışmada, tek basamaklı gerçek zamanlı kantitatif reverz transkripsiyon polimerize zincir reaksiyonu tekniği (qRTPCR) geliştirildi ve Türkiye’nin farklı coğrafik alanlarından elde edilen saha örneklerinde küçük ruminant vebası virusunun (PPRV) tespiti için karşılaştırıldı. Yerel PPRV (Tu00)’ nün dört genini (L, M, N ve P) hedefleyen primer ve problar ifade edilen virusun tüm genom dizini (AJ849636) temel alınarak tasarlandı. Çalışma kapsamında gerçekleştirilen tasarımlar ile her testin tespit sınırları, sırasıyla L geni için 7 kopya/µL RNA; P geni için 6 kopya/µL RNA; M geni için 10 kopya/µL RNA ve N geni için 700 kopya/µL RNA olarak bulundu. Bunun yanında, 45 saha örneği kullanılarak yapılan tanısal değerlendirmelerde ise virus genomunun tespit oranları sırasıyla L geni için %100 (45); M geni için %88.9 (40); P geni için %77.8 (35) ve N geni için %22.3 (10) olarak bulundu. Sonuç olarak, L geni dizaynı için bildirilen tek basamaklı gerçek zamanlı kantitatif reverz transkripsiyon polimerize zincir reaksiyonu deneyi (qRT-PCR) saha vakalarından elde edilen doku örneklerinde hızlı, spesifik ve hassas PPRV tespitini saplamıştır

Keywords

• Küçük ruminant vebası
• Gerçek zamanlı RT-PZR
• virus tespiti

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