Thompson Ryan, Stephenson Dominic, Sykes Hannah E, Perry John D, Stanforth Stephen P, Dean John R
Department of Applied Sciences, Northumbria University Ellison Building Newcastle upon Tyne NE1 8ST UK
Department of Microbiology, Freeman Hospital Newcastle upon Tyne NE7 7DN UK.
RSC Adv. 2020 Mar 12;10(18):10634-10645. doi: 10.1039/c9ra08386c. eCollection 2020 Mar 11.
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 and pathogens of the 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 ( 3-amino--(3-fluorophenyl)propanamide) that interacts with β-alanyl aminopeptidase to generate an exogenous volatile organic compound 3-fluoroaniline (LOD 0.02 μg mL; LOQ 0.06 μg mL). 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 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.
一种新颖、快速且灵敏的分析方法已被开发出来，并应用于105份来自囊性纤维化患者的痰液样本，其中包括5份肺移植后患者的样本。这种新方法专门用于测量β-丙氨酰氨基肽酶的活性，该酶活性是一些重要革兰氏阴性病原体的特征。与本研究相关的是 和 复合体的病原体，这两种病原体通常与呼吸道感染以及成年囊性纤维化患者发病率和死亡率增加有关。该分析方法涉及添加一种新型酶底物（3-氨基- -（3-氟苯基）丙酰胺），它与β-丙氨酰氨基肽酶相互作用生成一种外源性挥发性有机化合物3-氟苯胺（检测限0.02μg/mL；定量限0.06μg/mL）。通过顶空固相微萃取-气相色谱-质谱联用技术在痰液样本中测定3-氟苯胺的含量，其测定值高于计算定量限的20倍，然后将结果与标准培养方法以及使用基质辅助激光解吸电离飞行时间质谱进行细菌鉴定的结果进行比较。痰液样本仅孵育8小时后就有可能检测到3-氟苯胺，成功率为95%；24小时时成功率提高到100%，这完全在48小时的典型常规时间范围内。据我们所知，这是首次通过使用定制设计的底物释放可检测且独特的挥发性有机化合物来检测 的证明。极高的阴性预测值（本研究中为100%）意味着这样的检测方法可能适合作为尚未被这种病原体定植的患者的筛查技术。
