利用16SrRNA建立一种简单准确的无色杆菌种特异性鉴定方法。

IF 3.3 3区 医学 Q2 MICROBIOLOGY
Giulia Maria Saitta, Laura Veschetti, Rebecca Feletti, Angela Sandri, Marzia Boaretti, Paola Melotti, Maria Carelli, Maria M Lleò, Giovanni Malerba, Caterina Signoretto
{"title":"利用16SrRNA建立一种简单准确的无色杆菌种特异性鉴定方法。","authors":"Giulia Maria Saitta, Laura Veschetti, Rebecca Feletti, Angela Sandri, Marzia Boaretti, Paola Melotti, Maria Carelli, Maria M Lleò, Giovanni Malerba, Caterina Signoretto","doi":"10.3390/pathogens14030271","DOIUrl":null,"url":null,"abstract":"<p><p>The <i>Achromobacter</i> 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 <i>Achromobacter</i> 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: <i>A. xylosoxidans</i>, <i>A. dolens</i>, and <i>A. insuavis</i>. 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.</p>","PeriodicalId":19758,"journal":{"name":"Pathogens","volume":"14 3","pages":""},"PeriodicalIF":3.3000,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11945698/pdf/","citationCount":"0","resultStr":"{\"title\":\"Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of <i>Achromobacter</i> spp.\",\"authors\":\"Giulia Maria Saitta, Laura Veschetti, Rebecca Feletti, Angela Sandri, Marzia Boaretti, Paola Melotti, Maria Carelli, Maria M Lleò, Giovanni Malerba, Caterina Signoretto\",\"doi\":\"10.3390/pathogens14030271\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The <i>Achromobacter</i> 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 <i>Achromobacter</i> 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: <i>A. xylosoxidans</i>, <i>A. dolens</i>, and <i>A. insuavis</i>. 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.</p>\",\"PeriodicalId\":19758,\"journal\":{\"name\":\"Pathogens\",\"volume\":\"14 3\",\"pages\":\"\"},\"PeriodicalIF\":3.3000,\"publicationDate\":\"2025-03-12\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11945698/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Pathogens\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3390/pathogens14030271\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Pathogens","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3390/pathogens14030271","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0

摘要

无色杆菌属包括22种和不同的基因群。一些具有较高毒力或抗生素耐药性的物种更有可能引起囊性纤维化(CF)患者的慢性感染。目前的鉴定方法往往不能准确区分物种,或由于生物化学相似性导致错误鉴定。本研究旨在开发一种适用于临床诊断实验室的准确的物种水平鉴定qPCR方案。对不同色盲杆菌临床分离株进行全基因组测序,发现两个16S基因区域存在物种特异性单核苷酸多态性(snp)。基于这些snp,设计了两套引物和qPCR探针来生成独特的鉴定图谱。优化热谱图,并对一系列细菌DNA稀释液进行qPCR以确定检测限(LOD)。4种探针成功鉴定出3种:木犀草、dolens和insuavis。另外两个探针设计用于与公开可用序列无关的新基因型。检出限范围为0.005 pg/µL ~ 1 pg/µL。联合探针的灵敏度为100%,特异度为97.95% ~ 100%。该qPCR协议能够准确的物种鉴定,克服了当前方法的局限性,并代表了临床诊断的可靠工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of Achromobacter spp.

The Achromobacter 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 Achromobacter 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: A. xylosoxidans, A. dolens, and A. insuavis. 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.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Pathogens
Pathogens Medicine-Immunology and Allergy
CiteScore
6.40
自引率
8.10%
发文量
1285
审稿时长
17.75 days
期刊介绍: Pathogens (ISSN 2076-0817) publishes reviews, regular research papers and short notes on all aspects of pathogens and pathogen-host interactions. There is no restriction on the length of the papers. Our aim is to encourage scientists to publish their experimental and theoretical research in as much detail as possible. Full experimental and/or methodical details must be provided for research articles.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信