Isola Daniela, Pardini Manuela, Varaine Francis, Niemann Stefan, Rüsch-Gerdes Sabine, Fattorini Lanfranco, Orefici Graziella, Meacci Francesca, Trappetti Claudia, Rinaldo Oggioni Marco, Orrù Germano
Dipartimento di Chirurgia e Scienze Odontostomatologiche, Università di Cagliari, Cagliari, Italy.
J Microbiol Methods. 2005 Jul;62(1):113-20. doi: 10.1016/j.mimet.2005.02.004.
Ethambutol (EMB) is in use worldwide as a first-line anti-tuberculosis drug and substitutions in codon 306 of the embB gene are the most common mutations found in EMB resistant Mycobacterium tuberculosis (MTB) strains. Pyrosequencing is a real time sequencing method able to rapidly detect mutations in a large number of samples. Using this technique we analyzed, in parallel with conventional sequencing, a 24 bp region of the embB gene of 28 MTB clinical isolates. Pyrosequencing efficiently identified all embB306 mutations, detecting three different single-base substitutions leading to 2 amino acid changes (Met to Val or Ile). Mutated embB alleles were detected in 2 multidrug-resistant (MDR) EMB-susceptible strains. Overall, our results demonstrated that the Pyrosequencing method efficiently recognizes mutations in embB in a very short time and represents a valid molecular method to detect mutations in the MTB embB306 region.
乙胺丁醇（EMB）作为一线抗结核药物在全球范围内使用，embB基因第306位密码子的替代是耐乙胺丁醇结核分枝杆菌（MTB）菌株中最常见的突变。焦磷酸测序是一种能够快速检测大量样本中突变的实时测序方法。我们使用该技术与传统测序并行分析了28株MTB临床分离株embB基因的一个24bp区域。焦磷酸测序有效地鉴定了所有embB306突变，检测到导致2个氨基酸变化（甲硫氨酸变为缬氨酸或异亮氨酸）的3种不同单碱基替代。在2株耐多药（MDR）但对乙胺丁醇敏感的菌株中检测到了突变的embB等位基因。总体而言，我们的结果表明，焦磷酸测序方法能在极短时间内有效识别embB中的突变，是检测MTB embB306区域突变的一种有效分子方法。
