Alkalen Fosfataz-Bazli Virüs Odağı (ALPspot) Saptama Testi Geliştirilmesi: Enfeksiyoz Rotavirüs Titresinin Belirlenmesi

ovine rotavirus (BRV), buzağılarda neonatal ishalin başlıca nedenidir ve süt ve hayvancılık üretiminde önemli ekonomik kayıplara neden olur. Doku kültürü bulaşıcı dozu (TCID50), neredeyse bir asır önce virüs titrelerini ölçmek için tasarlanmış sitopatik etki (CPE) tabanlı bir mikroskobik inceleme testidir. Virüsleri çoğaltırken hücre kültürlerinde CPE'leri indüklemediğinde virüs titrelerini belirlemek zordur ve daha doğru ve güvenilir yöntemlerin geliştirilmesi gerekir. Yeni doğan buzağılardan dışkı örnekleri toplandı ve BRV'yi saptamak için bir RT-PCR testi yapıldı. BRV suşlarını fekal süspansiyonlardan izole etmek için hücre kültür virüs izolasyon yöntemi uygulandı ve ELISA ile doğrulandı. Alkalin fosfataz temelli virüs odak (ALPSpot) testini geliştirmek ve doğrulamak için izole edilen BRV suşları kullanıldı. Alkalin fosfatazla konjuge antikorları katalize eden kromojenik bir substratın eklenmesinden sonra virüs odakları görünür hale getirildi. Tek adımlı RT-PCR testi ile toplam 17 dışkı örneği Rotavirüs için pozitif test edildi. Hücre kültürü virüs izolasyon çalışmaları yoluyla 6 BRV izolatı başarıyla izole edildi ve ELISA'da pozitif test edildi (Cut-off: 0.247). Test duyarlılığı, TCID50 titre yöntemine kıyasla daha yüksek bir korelasyon oranı (R2=0.9833) ile ölçülmüştür ve virüs titresi açısından TCID50 ve ALPSpot yöntemi arasında istatistiki anlamlı bir fark bulunmadı. Ayrıca ALPSpot, TCID50 ile karşılaştırıldığında en düşük virüs titresini dahi tespit edebildi. ALPSpot testi, duyarlı hücre kültürlerinde BRV titrelerini ölçmek için indüklenmiş CPE gerektirmeyen güvenilir ve doğru bir yöntemdir ve küçük modifikasyonlarla koruma çalışmalarında aşı ile indüklenen veya pasif olarak aktarılan nötralize edici antikorları belirlemek için kullanılabilir. Bu çalışmada BRV titresi tespiti için ALPSpot saptama testi geliştirilerek optimize edilmesi tarif edildi.

The Development of an Alkaline Phosphatase-Based Virus Spot (ALPSpot) Detection Assay: Determination of Infectious Rotavirus Titer

Bovine rotavirus (BRV) is major cause of neonatal diarrhea in calves and cause substantial economic loss in dairy and livestock production. The tissue culture infectious dose (TCID50) is a cytopathic effect (CPE)-based microscopic examination assay designed to measure virus titers almost a century ago. It is difficult to determine the virus titers when replicating viruses do not induce CPEs in cell cultures and more accurate and reliable methods need to be developed. Fecal samples were obtained from newborn calves and a RT-PCR assay was performed to detect BRV. A cell culture isolation method was performed to isolate BRV strains in fecal suspensions and confirmed by ELISA. All BRV strains were employed to develop and validate ALPSpot assay. Spots become visible after the addition of a chromogenic substrate that catalyzes alkaline phosphatase- conjugated antibodies. A total of 17 fecal samples tested positive for BRV in one-step RT-PCR. Only 6 BRV isolates were successfully rescued through cell culture virus isolation studies and tested positive in ELISA (Cut- off: 0.247). Assay sensitivity was measured with a higher correlation rate (R2=0.9833) when compared to the TCID50 titer method and there are no significant differences between TCID50 and ALPSpot methods in terms of virus titer. In addition, ALPSpot also determines the lowest virus titer when compared to TCID50. In this study, an alkaline phosphatase spot (ALPSpot) detection assay has been optimized to determine BRV titers.

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