Saitta Giulia Maria, Veschetti Laura, Feletti Rebecca, Sandri Angela, Boaretti Marzia, Melotti Paola, Carelli Maria, Lleò Maria M, Malerba Giovanni, Signoretto Caterina
Diagnostic and Public Health Department, University of Verona, 37134 Verona, Italy.
Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 20132 Milano, Italy.
Pathogens. 2025 Mar 12;14(3):271. doi: 10.3390/pathogens14030271.
The genus comprises 22 species and various genogroups. Some species with higher virulence or antibiotic resistance are more likely to cause chronic infections in people with cystic fibrosis (CF). Current identification methods often fail to accurately distinguish between the species or result in misidentifications due to biochemical similarities. This study aims to develop an accurate qPCR protocol for species-level identification that is applicable in clinical diagnostic laboratories. Whole-genome sequencing of clinical isolates from different species identified species-specific single-nucleotide polymorphisms (SNPs) in two 16S gene regions. Based on these SNPs, two sets of primers and qPCR probes were designed to generate unique identification profiles. Thermal profiles were optimized, and qPCR was performed on serial bacterial DNA dilutions to determine the detection limit (LOD). Four probes successfully identified three species: , , and . Two additional probes were designed for novel genotypes unrelated to publicly available sequences. The LOD ranged from 0.005 pg/µL to 1 pg/µL. Combined probes achieved 100% sensitivity, with specificity ranging from 97.95% to 100%. This qPCR protocol enables accurate species identification, overcoming the limitations of current methods, and represents a reliable tool for clinical diagnostics.
该属包括22个物种和各种基因群。一些具有较高毒力或抗生素耐药性的物种更有可能在囊性纤维化（CF）患者中引起慢性感染。目前的鉴定方法往往无法准确区分这些物种，或者由于生化相似性而导致错误鉴定。本研究旨在开发一种适用于临床诊断实验室的、用于物种水平鉴定的准确qPCR方案。对来自不同物种的临床分离株进行全基因组测序，在两个16S基因区域鉴定出物种特异性单核苷酸多态性（SNP）。基于这些SNP，设计了两组引物和qPCR探针，以生成独特的鉴定图谱。优化了热循环条件，并对系列细菌DNA稀释液进行qPCR以确定检测限（LOD）。四个探针成功鉴定出三个物种： 、 和 。另外设计了两个探针用于与公开可用序列无关的新基因型。LOD范围为0.005 pg/µL至1 pg/µL。组合探针的灵敏度达到100%，特异性范围为97.95%至100%。这种qPCR方案能够实现准确的物种鉴定，克服了当前方法的局限性，是临床诊断的可靠工具。
