Iahtasham KHAN, Shahzad ALI, Mandy ELSCHNER, Katja MERTENS, Falk MELZER, Haroon AHMED, Muhammad SAQIB, Muhammad IJAZ, Mayada GWIDA, Heinrich NEUBAUER

Evaluation of Analytical-Sensitivity and -Specificity of a Commercially Available One-Step Real-Time Polymerase Chain Reaction Assay Kit for the Detection of Burkholderia mallei

Burkholderia mallei (B. mallei) ruamın etiyolojik ajanı olup, hastalık tek tırnaklıların oldukça enfeksiyöz ve bulaşıcı bir hastalığıdır. B. mallei'nin hızlı ve doğru tespiti ve ruamın diagnozu hem insanlar hem de hayvanlar için önemlidir. Bu çalışma, B. mallei'nin tespitinde ticari olarak satılan tek-basamaklı gerçek-zamanlı (RT) PCR kitinin (Genekam Biotechnology AG, Almanya) analitik özgünlüğü ve özgüllüğünün değerlendirilmesi amacıyla yapılmıştır. Daha önceden yayınlanmış olan fliP RT-PCR protokolü ile mevcut test edilen kitin özgünlüğü ve özgüllüğünün karşılaştırılması gerçek-zamanlı PCR, Mx3000P TM (Stratagene®, Kanada) ile yapıldı. Diagnostik parametreler 20 B. mallei, 20 B. pseudomallei, 15 B. mallei alakalı ve 10 klinik olarak ilgili Burkholderia olmayan türler ile değerlendirilirdi. Mx3000PTM platformunda tek basamaklı RT-PCR uygulayarak tespit etme limiti (LOD) 10 fg olarak belirlendi. Mx3000PTM platformu kullanılarak uygulanan fliP RT-PCR ile LOD 100 fg olarak belirlendi. Tek basamaklı RT-PCR'ın B. mallei suşları için spesifik olduğu sonucuna varıldı. Tek basamaklı RT-PCR kiti, kültürden B. mallei'nin spesifik olarak identifiye edilmesi ve belirlenmesinde kullanılabilecek hızlı geri dönüş zamanına sahip güçlü, hızlı, güvenilir, spesifik ve hassas bir üründür

Burkholderia mallei’nin Tespitinde Ticari Tek-Basamaklı Gerçek-Zamanlı Polimeraz Zincir Reaksiyon Kitinin Analitik Özgünlüğü ve Özgüllüğünün Değerlendirilmesi

Burkholderia mallei (B. mallei) is the etiological agent of glanders, a highly infectious and zoonotic disease of solipeds. Prompt and accurate detection of B. mallei and diagnosis of glanders is important for both, humans and animals. The present study was designed to evaluate the analytical sensitivity and specificity of a commercially available one-step real-time (RT) PCR assay kit (Genekam Biotechnology AG, Germany) for the detection of B. mallei. Furthermore, the comparative evaluation of the analytical sensitivity of an already published fliP RT-PCR protocol and this kit was made using a real-time PCR (RT-PCR) platform: the Mx3000P TM (Stratagene®, Canada). Diagnostic parameters were assessed with a panel of 20 B. mallei, 20 B. pseudomallei, 15 B. mallei related and 10 clinically relevant non-Burkholderia species. Using the one-step RT-PCR on the Mx3000PTM platform, the limit of detection (LOD) was determined as 10 fg. Applying a modified fliP RT-PCR using the Mx3000P platform resulted in a LOD of 100 fg. Authors conclude that the one-step RT-PCR kit is specific for DNA of B. mallei strains. The one-step RT-PCR assay kit is a robust, rapid, reliable, specific and sensitive tool with a fast turnaround time for the specific identification and detection of B. mallei from culture material

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