Sheena Conforti , Pablo Rossi Orts , Manu Tamminen , Timothy R. Julian
{"title":"利用数字多重连接法高通量多重检测大肠杆菌耐药基因和毒力因子。","authors":"Sheena Conforti , Pablo Rossi Orts , Manu Tamminen , Timothy R. Julian","doi":"10.1016/j.jmoldx.2025.03.003","DOIUrl":null,"url":null,"abstract":"<div><div><em>Escherichia coli</em> causes >400,000 annual deaths in children aged <5 years worldwide, with morbidity and mortality exacerbated by antimicrobial-resistant strains. A high-throughput multiplexing assay called digital multiplex ligation assay (dMLA) was developed to detect simultaneously 43 priority genes in <em>E. coli</em> related to the following: antibiotic resistance (<em>n</em> = 19), virulence factors (<em>n</em> = 16), and phylogroup markers (<em>n</em> = 6) with controls (<em>uidA</em>, <em>gapdh</em>). Genes are detected via PCR amplification of adjacent probe pairs that ligate in the presence of target gene-specific DNA, followed by sequencing of amplicons on short-read sequencers. The assay was tested in technical replicates on 63 synthetic DNA controls, and applied to 58 <em>E. coli</em>, 2 <em>Staphylococcus aureus</em>, 2 <em>Klebsiella pneumoniae</em>, 1 <em>Klebsiella oxytoca</em>, 1 <em>Vibrio cholera</em>, 1 <em>Pseudomonas lurida</em>, and 1 <em>Salmonella enterica</em> isolates in duplicate. Whole-genome sequencing was used to assess specificity and sensitivity. dMLA showed 100% sensitivity and >99.9% specificity and balanced accuracy on synthetic DNA. Balanced accuracy, calculated as the average of sensitivity and specificity, accounts for imbalanced data sets where negative outcomes are significantly more prevalent than positive ones. dMLA achieved a balanced accuracy of 90% for bacterial isolates. The results underline dMLA's effectiveness in high-throughput characterization of <em>E. coli</em> libraries for antimicrobial resistance genes and virulence factors, leveraging sequencing for massively parallel multiplexing of gene regions on multiple samples simultaneously, and are extendable to targets beyond <em>E. coli.</em></div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 6","pages":"Pages 511-524"},"PeriodicalIF":3.4000,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"High-Throughput Multiplex Detection of Antibiotic-Resistant Genes and Virulence Factors in Escherichia coli Using Digital Multiplex Ligation Assay\",\"authors\":\"Sheena Conforti , Pablo Rossi Orts , Manu Tamminen , Timothy R. Julian\",\"doi\":\"10.1016/j.jmoldx.2025.03.003\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div><em>Escherichia coli</em> causes >400,000 annual deaths in children aged <5 years worldwide, with morbidity and mortality exacerbated by antimicrobial-resistant strains. A high-throughput multiplexing assay called digital multiplex ligation assay (dMLA) was developed to detect simultaneously 43 priority genes in <em>E. coli</em> related to the following: antibiotic resistance (<em>n</em> = 19), virulence factors (<em>n</em> = 16), and phylogroup markers (<em>n</em> = 6) with controls (<em>uidA</em>, <em>gapdh</em>). Genes are detected via PCR amplification of adjacent probe pairs that ligate in the presence of target gene-specific DNA, followed by sequencing of amplicons on short-read sequencers. The assay was tested in technical replicates on 63 synthetic DNA controls, and applied to 58 <em>E. coli</em>, 2 <em>Staphylococcus aureus</em>, 2 <em>Klebsiella pneumoniae</em>, 1 <em>Klebsiella oxytoca</em>, 1 <em>Vibrio cholera</em>, 1 <em>Pseudomonas lurida</em>, and 1 <em>Salmonella enterica</em> isolates in duplicate. Whole-genome sequencing was used to assess specificity and sensitivity. dMLA showed 100% sensitivity and >99.9% specificity and balanced accuracy on synthetic DNA. Balanced accuracy, calculated as the average of sensitivity and specificity, accounts for imbalanced data sets where negative outcomes are significantly more prevalent than positive ones. dMLA achieved a balanced accuracy of 90% for bacterial isolates. The results underline dMLA's effectiveness in high-throughput characterization of <em>E. coli</em> libraries for antimicrobial resistance genes and virulence factors, leveraging sequencing for massively parallel multiplexing of gene regions on multiple samples simultaneously, and are extendable to targets beyond <em>E. coli.</em></div></div>\",\"PeriodicalId\":50128,\"journal\":{\"name\":\"Journal of Molecular Diagnostics\",\"volume\":\"27 6\",\"pages\":\"Pages 511-524\"},\"PeriodicalIF\":3.4000,\"publicationDate\":\"2025-04-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Molecular Diagnostics\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1525157825000832\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"PATHOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Molecular Diagnostics","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1525157825000832","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PATHOLOGY","Score":null,"Total":0}
High-Throughput Multiplex Detection of Antibiotic-Resistant Genes and Virulence Factors in Escherichia coli Using Digital Multiplex Ligation Assay
Escherichia coli causes >400,000 annual deaths in children aged <5 years worldwide, with morbidity and mortality exacerbated by antimicrobial-resistant strains. A high-throughput multiplexing assay called digital multiplex ligation assay (dMLA) was developed to detect simultaneously 43 priority genes in E. coli related to the following: antibiotic resistance (n = 19), virulence factors (n = 16), and phylogroup markers (n = 6) with controls (uidA, gapdh). Genes are detected via PCR amplification of adjacent probe pairs that ligate in the presence of target gene-specific DNA, followed by sequencing of amplicons on short-read sequencers. The assay was tested in technical replicates on 63 synthetic DNA controls, and applied to 58 E. coli, 2 Staphylococcus aureus, 2 Klebsiella pneumoniae, 1 Klebsiella oxytoca, 1 Vibrio cholera, 1 Pseudomonas lurida, and 1 Salmonella enterica isolates in duplicate. Whole-genome sequencing was used to assess specificity and sensitivity. dMLA showed 100% sensitivity and >99.9% specificity and balanced accuracy on synthetic DNA. Balanced accuracy, calculated as the average of sensitivity and specificity, accounts for imbalanced data sets where negative outcomes are significantly more prevalent than positive ones. dMLA achieved a balanced accuracy of 90% for bacterial isolates. The results underline dMLA's effectiveness in high-throughput characterization of E. coli libraries for antimicrobial resistance genes and virulence factors, leveraging sequencing for massively parallel multiplexing of gene regions on multiple samples simultaneously, and are extendable to targets beyond E. coli.
期刊介绍:
The Journal of Molecular Diagnostics, the official publication of the Association for Molecular Pathology (AMP), co-owned by the American Society for Investigative Pathology (ASIP), seeks to publish high quality original papers on scientific advances in the translation and validation of molecular discoveries in medicine into the clinical diagnostic setting, and the description and application of technological advances in the field of molecular diagnostic medicine. The editors welcome for review articles that contain: novel discoveries or clinicopathologic correlations including studies in oncology, infectious diseases, inherited diseases, predisposition to disease, clinical informatics, or the description of polymorphisms linked to disease states or normal variations; the application of diagnostic methodologies in clinical trials; or the development of new or improved molecular methods which may be applied to diagnosis or monitoring of disease or disease predisposition.