Real time BKV PCR testinin analitik performansı

Öz

Amaç: BKV, çocukluk çağında genellikle asemptomatik olarak geçirilen ve özellikle böbreklerde olmak üzere perifik kan ve beyinde latent olarak kalabilen bir virustür. BVK’nın aktivasyonu immünsuprasyonu olan hastalarda hemorajik veya hemorajik olmayan sistit, ürostenoz ve nefropatiye neden olabilir. Özellikle transplant alıcılarında nefropati sıklığı %5’lere kadar ulaşabilmekte ve transplante edilen organın %30-60 erken kaybı ve kötü bir klinik seyirle sonuçlanabilmektedir. BKV viral yükünün serum ve idrar örneklerinde polimeraz zincir reaksiyonu PZR ile kantitasyonu, erken tanı ve tedavinin yönetiminde önemli rol oynamaktadır. Real time PZR; klasik PZR’ye göre daha duyarlı, kantitasyon yapabilen, kontaminasyon riski az ve sonuçlanma süresinin kısa olması nedeniyle son yıllarda yaygın olarak kullanılmaktadır. Bu çalışmamızda BKV tanısı için laboratuvarımızda tasarlanan kantitatif real time BKV PZR testinin analitik performansının değerlendirilmesi amaçlanmıştır. Yöntem: BKV plazmidinden ATCC 45025 standardlar hazırlanmıştır. 15 x 107 kopya/ml’den 3 x 101 kopya/ml’ye kadar seri dilüsyonlar içeren BKV plazmidinin spektrofotometrik ölçümü yapılmıştır. Amplifikasyon reaksiyonları için BKV VP1 gen bölgesine uygun, 5’ ucu 6-karboksifloreseinle FAM , 3’ ucu 6-karboksitetrametilrodamin TAMRA ile işaretli primerler daha önce belirtildiği gibi kullanılmıştırBulgular: Çalışmada testin analitik performansının değerlendirilmesi için analitik duyarlılık, özgüllük, doğrusallık, doğruluk ve kesinlik parametreleri belirlenmiştir. Testin analitik sensitivitesi 15 x 102 kopya/ml ve saptama limiti 5 x 102 kopya/ml olarak belirlenmiştir. 15 x 107 kopya/ml ile 15 x 101 kopya/ ml konsantrasyonlar arasında çalışılan dilüsyonların standard deviasyonu SD 0,02 ile 0,644 arasında, varyasyon katsayısı CV ise %0,79 ile %11,47 arasında değişmiştir. 19 dış kalite kontrol örneğinin sonuçları ilgili programla %100 uyumludur. Testimiz 15 x 102 kopya/ml ile 15 x 108 kopya/ml arasında lineer bir seyir göstermiştir. Özgüllüğü ise %100 olarak belirlenmiştir. Ayrıca dış kalite kontrol programı ile kalite kontrol örneklerinin sonuçları çok yakın uygunluktadır. Sonuç: Elde edilen sonuçlarımız doğrultusunda laboratuvar tasarımlı real time PCR protokolümüz; duyarlı, özgül, kesin ve geniş dinamik aralıklı tekraralanabilirliği olan bir test olarak bulunmuştur.

Anahtar Kelimeler

• Analitik performans, Real Time BKV PCR, Validasyon deneyleri

The analytical performance of a real time BKV PCR assay

Öz

Objective: BKV is a virus usually undergone asymptomatically in the childhood and can remain latent in the peripheral blood, brain and especially in kidneys. Reactivation of BKV under immunosuppression can cause diseases like interstitial nephritis, haemorrhagic or non- haemorrhagic cystitis, ureterostenosis and nephropathy. Especially in transplant recipients nephropathy frequency can reach 5% and can be the cause of premature loss 30-60% of transplanted organs and poor outcome. Quantification of BKV viral load in urine and serum with real time polymerase chain reaction PCR plays important role for early diagnosis and management of the therapy. Since the Real time PCR assay is more sensitive than classical PCR, can do quantification and have a less risk of contamination and short turn-around time. The aim of our study was to evaluate the analytical performance of a real time quantitative BKV PCR assay which was developed in our laboratory. Method: Standards were prepared from BKV plasmid ATCC 45025 . BKV plasmid that contained 15 x 107 copies/ ml to 3 x 101 copies/ml serial dilutions was measured by spectrofotometery. Primers for BKV VP1 gene and dual labelled probe at the 5’ end with 6-carboxyfluoresceine FAM and the 3’ end with 6-carboxytetramethylrhodamine TAMRA as described previously were used for the amplification reactions. Results: To evaluate the analytical performance of the assay; analytical sensitivity, specificity, linearity, accuracy and precision was determined. The analytical sensitivity and the limit of detection of the assay were found 15 x 102 copies/ml and 5 x 102 copies/ ml, respectively. Standard deviation SD of dilutions varied from 0.02 to 0.644 and CV varied from 0.79% to 11.47% between 15 x 107 to 15 x 101 copies/ml concentrations. Nineteen proficiency samples results of quality control program were in close agreement 100% . The assay demonstrated a linear range from 15 x 102 to 15 x 108 copies/ml. Specificity of the assay was found 100%. In addition proficiency samples results of the external quality control program were in close agreement. Conclusion: According to our results the real time PCR protocol of BKV developed in our laboratory was found sensitive, specific, precise and reproducible with a broad dynamic range

Anahtar Kelimeler

• Analytical performance, Real Time BKV PCR, validation experiments

Kaynakça

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