Phương pháp này là cực kỳ nhạy cảm. Khi rRNA được sử dụng như một mục tiêu, khoảng bản sao có thể có mặt trên một tế bào. Hơn nữa, hàng trăm bản sao RNA được tạo ra trong chu kỳ, mỗi khuếch đại | V The Basic Kit amplification module for the detection of Candida spp. fungal RNA contamination of kit components Annemarie Borst Eijkman-Winkler Institute University Medical Center Utrecht the Netherlands The Basic Kit amplification module Nucleic Acid Sequence-Based Amplification NASBA is an isothermal RNA amplification method based on the simultaneous action of three enzymes Avian Myeloblastosis Virus Reverse Transcriptase AMV-RT RNase H and T7 RNA polymerase1. The method is extremely sensitive. When rRNA is used as a target as many as 10 000 copies can be present per cell. Furthermore hundreds of RNA copies are generated in each amplification cycle each of which serve as a target for the next round in comparison with PCR only two copies are generated in each cycle . This results in a large amount of product in a short period of time. NASBA was successfully used in our laboratory for the detection of Candida spp. in blood and blood cultures2 3. Primers and probes for the detection of several Candida spp. were developed and used in an in-house NASBA assay3. Yeast RNA was extracted by using RNAzol Campro Scientific Veenendaal the Netherlands and amplification products were detected using the Basic Kit electrochemiluminescence ECL detection module Organon Teknika Boxtel the Netherlands 2. The aim of this study was to replace our in-house NASBA assay by the Basic Kit amplification module Organon Teknika . The Basic Kit amplification module contains a reagent sphere comprised of . dNTP s and NTP s reagent sphere diluent a separate stock of KCl for optimization of the assay enzyme mix and NASBA-water. The primers are not included but have to be designed by the user in our case we could use the primers from our in-house NASBA assay . We spiked a mixture of blood from a healthy volunteer and aerobic blood culture medium BacT Alert FAN medium Organon Teknika with a 10-fold dilution of Candida albicans cfu. RNA was extracted as described2. After amplification using the
