Molecular techniques for clinical diagnostic bacteriology

Moleküler yöntemler enfeksiyon hastalıkların patogenezinin ve epidemiyolojisinin anlaşılmasında çok önemli katkıda bulunmuştur. Polimeraz zincir reaksiyonu (PZR), en yaygın kullanılan hedef nükleik asit amplifikasyon yöntemidir. Bu yöntem ile tek bir nükleik asidin kopyası çok kısa bir süre içinde 107 kereden fazla çoğalır. Gerçek zamanlı PZR, sekanslama tekniği ve kütle spektrofotometresi gibi yeni teknolojiler klinik mikrobiyoloji laboratuvarında birçok uygulama alanı kazanmıştır. Gerçek zamanlı tekniklerin en büyük etkisi viroloji alanında olmuştur ve bu tekniklerin klinik örneklerde çeşitli virüslerin tespiti, kantitatif viral yükleri ve antiviral tedaviye yanıtı izlemek için kullanılmıştır. Bakteriyoloji alanında bakteriyel patojenler ve/veya antibiyotik direnç genlerinin hızlı tanımlanması, antibiyotiklerin uygun kullanımını sağlar, hastanede kalış süresini kısaltır ve dirençli suşların gelişme potansiyelini azaltır. Neisseria gonorrhoeae ve Chlamydia trachomatis tespiti için PZR teknolojisi kullanan ticari kitler geliştirilmiştir. Nükleik asit amplifikasyon testleri klinik örneklerde Mycobacterium tuberculosis kompleksinin doğrudan tespitinde kullanılabilir. Bazı klinik laboratuvarlar, klinik örneklerden M. tuberculosisi tespit etmek için PZR deneylerine dayalı kendi in-house deneylerini geliştirmiştir. Bazı çalışmalar hazır kitleri ve in-house yöntemleri karşılaştırmıştır ve benzer sonuçlar bulmuştur. In-house geliştirilen yöntemlere ek olarak bazı ticari amplifikasyon kitleri yaygın olarak kullanılmaktadır. Bu bölümde güncel DNA yöntemine dayalı moleküler tekniklerin temel ilkelerini ve klinik bakteriyoloji için uygulamaları açıklanmıştır.

Klinik bakteriyoloji tanısında moleküler teknikler

Molecular methods have contributed tremendously to the understanding of the pathogenesis and epidemiology of infectious diseases. The polymerase chain reaction (PCR) is the most widely used target nucleic acid amplification method. By this method, a single copy of a nucleic acid is multiplied to more than 107 times within a very short period. New technologies such as real-time PCR, sequencing and mass spectrophotometry have been described and have many applications in a clinical microbiology laboratory. The greatest impact of real-time assays was in the field of virology where they have been used to detect rapidly a range of viruses in human specimens and to monitor quantitatively viral loads and response to antiviral therapy. In bacteriology they are used for rapid detection of bacterial pathogens and/or antibiotic resistance genes can help to ensure the appropriate use of antibiotics, reduce the duration of hospital stay and minimize the potential for resistant strains of bacteria to emerge. Commercial kits employing PCR technology for detection of Neisseria gonorrhoeae and Chlamydia trachomatis have been developed. Nucleic acid amplification tests can be used directly to identify Mycobacterium tuberculosis complex in clinical specimens. Some clinical laboratories have developed their in-house assays based on PCR assays to detect M. tuberculosis in clinical specimens. Some studies have compared the kits and in-house methods and have found similar results. In addition to in-house developed assays, there are commercial amplification tests some of them that are widely used. This chapter describes the basic principles and applications of recently DNA-based molecular techniques for the clinical bacteriology.

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