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首页> 外文期刊>RSC Advances >Detection of β-alanyl aminopeptidase as a biomarker for Pseudomonas aeruginosa in the sputum of patients with cystic fibrosis using exogenous volatile organic compound evolution
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Detection of β-alanyl aminopeptidase as a biomarker for Pseudomonas aeruginosa in the sputum of patients with cystic fibrosis using exogenous volatile organic compound evolution

机译：使用外源性挥发性有机化合物进化的囊性纤维化患者在囊性纤维化患者痰中的β-奥烷氨基肽酶作为假单胞菌的生物标志物

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A novel, rapid and sensitive analytical method has been developed and applied to 105 sputum samples from patients with cystic fibrosis, including 5 samples from post-lung transplant patients. This new method is specifically targeted to measure β-alanyl aminopeptidase activity which is characteristic of some important Gram-negative pathogens. Of relevance to this study are Pseudomonas aeruginosa and pathogens of the Burkholderia cepacia complex both of which are commonly associated with respiratory infections as well as increased morbidity and mortality in adult cystic fibrosis patients. The analytical method involves the addition of a novel enzyme substrate ( i.e. 3-amino- N -(3-fluorophenyl)propanamide) that interacts with β-alanyl aminopeptidase to generate an exogenous volatile organic compound 3-fluoroaniline (LOD 0.02 μg mL ~(?1) ; LOQ 0.06 μg mL ~(?1) ). 3-Fluoroaniline was determined at 20 times above its calculated limit of quantification in the sputum samples by HS-SPME-GC-MS and then the results compared with standard culture methods and bacterial identification using MALDI-TOF-MS. Detection of 3-fluoroaniline was possible after only 8 h incubation of the sputum samples with a 95% success rate; this increased to 100% at 24 h which was well within the typical routine timeframe of 48 h. To our knowledge, this is the first demonstration of detection of P. aeruginosa by use of a custom-designed substrate to liberate a detectable and unique VOC. The very high negative predictive value (100% in this study) means such an assay could be appropriate as a screening technique for patients who are not yet colonized by this pathogen.
机译：已经开发了一种新颖的，快速敏感的分析方法，并应用于患有囊性纤维化患者的105个痰样品，其中来自肺后移植患者的5个样品。该新方法特异性地靶向测量β-丙氨酸氨基肽酶活性，其是一些重要革兰氏阴性病原体的特征。与本研究的相关性是假单胞菌铜绿假单胞菌和伯克德列风瘢痕疙瘩的病原体，两者都与呼吸道感染通常有关，以及成人囊性纤维化患者的发病率和死亡率增加。分析方法涉及添加新的酶底物（即3-氨基 - （3-氨基 - （3-氟苯基）丙酰胺），其与β-丙氨酸氨基肽酶相互作用，得到外源性挥发性有机化合物3-氟二苯胺（LOD0.02μgmL〜（ ？1）; LOQ0.06μgmL〜（？1））。通过HS-SPME-GC-MS在痰液样品中计算出的3-氟苯胺，在其计算的痰液中的定量限制，然后与使用MALDI-TOF-MS的标准培养方法和细菌鉴定进行比较。在仅8小时的成功率为95％的痰样品中仅孵育3-氟硅氧烷的检测是可能的。在24小时的情况下，这在48小时的典型常规时间范围内增加到100％。据我们所知，这是通过使用定制设计的基材来解除P.铜绿假单胞菌的第一次证明以释放可检测和独特的VOC。非常高的负预测值（本研究中100％）是指这种测定可以作为尚未通过该病原体殖民殖民的患者的筛选技术。

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