Validation of microbial source tracking markers through PCR-based molecular analysis and microbial genome database

IF 3.6 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Current Research in Biotechnology Pub Date : 2025-01-01 DOI:10.1016/j.crbiot.2025.100304

Rifat Zubair Ahmed , Ashraful Islam , Tiantian Tian , Zhe Tian , Yu Zhang , Min Yang

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引用次数: 0

Abstract

Microbial Source Tracking (MST) uses molecular markers targeting host-associated gut microorganisms to identify fecal pollution. However, MST faces significant challenges in fecal source identification, particularly due to the markers’ poor specificity and shared genomic areas among microorganisms from different host sources. This study addresses these challenges by using host-specific Escherichia coli genetic markers, originally developed through a novel, library-independent approach, to detect sources of fecal pollution. A total of 563 E.coli isolates from chicken, cow, and pig feces were isolated and assessed by nine reported host-associated E. coli genetic markers (Chicken: CH7, CH9, CH12, CH13; Cow: CO2, CO3; Pig: P1, P3, P4) through PCR. Marker possession patterns, sensitivity, specificity, and accuracy were calculated. The NCBI Microbial Genome database was searched for sequences homologous to genome regions of studied genetic markers and evaluated by finding the percentage of host sources and sequence location in the genome. Homology evaluation with binary PCR results was used to predict the best-performing marker. PCR results exhibited that the most effective markers were chicken CH7 (67% sensitivity, 77.9% specificity, 74.4% accuracy) and CH9 (55% sensitivity, 99.4% specificity, 84.7% accuracy). However, a homology search in the database narrowed the selection of the top-performing marker to CH7, which showed homology with E.coli from chicken hosts, while other markers exhibited higher homology with E.coli from Humans. Furthermore, sequences from the database homologous to the CH9 and CO2 markers were found on a plasmid, while those for CH12, CO3, P1, and P4 were on the chromosome, and CH7, CH13, and P3 were on both. This study highlights the critical need for integrated approaches to assess molecular markers in MST assays, emphasizing their significance in advancing research within the field.

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通过基于pcr的分子分析和微生物基因组数据库验证微生物源跟踪标记

微生物源追踪技术（MST）是一种利用分子标记靶向宿主相关肠道微生物来识别粪便污染的技术。然而，MST在粪便来源鉴定方面面临着重大挑战，特别是由于标记的特异性较差，并且来自不同宿主来源的微生物具有相同的基因组区域。本研究通过使用宿主特异性大肠杆菌遗传标记来解决这些挑战，该标记最初是通过一种新颖的、独立于文库的方法开发的，用于检测粪便污染源。从鸡、牛和猪粪便中分离出563株大肠杆菌，并利用9个已报道的宿主相关大肠杆菌遗传标记(鸡：CH7、CH9、CH12、CH13；牛：CO2， CO3；猪：P1， P3, P4)。计算标记物占有模式、敏感性、特异性和准确性。在NCBI微生物基因组数据库中搜索与所研究遗传标记的基因组区域同源的序列，并通过寻找宿主来源的百分比和序列在基因组中的位置进行评估。用二元PCR结果进行同源性评价，预测最佳标记。PCR结果显示，鸡CH7（敏感性67%，特异性77.9%，准确性74.4%）和CH9（敏感性55%，特异性99.4%，准确性84.7%）是最有效的标记。然而，在数据库中进行同源性搜索，将选择的最佳标记缩小到CH7，该标记与鸡宿主大肠杆菌具有同源性，而其他标记与人类大肠杆菌具有较高的同源性。此外，在一个质粒上发现了数据库中与CH9和CO2标记同源的序列，而CH12、CO3、P1和P4标记在染色体上，CH7、CH13和P3标记在两条染色体上。这项研究强调了在MST检测中评估分子标记的综合方法的迫切需要，强调了它们在推进该领域研究中的重要性。

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来源期刊

Current Research in Biotechnology Biochemistry, Genetics and Molecular Biology-Biotechnology

CiteScore

6.70

自引率

3.60%

发文量

审稿时长

38 days

期刊介绍： Current Research in Biotechnology (CRBIOT) is a new primary research, gold open access journal from Elsevier. CRBIOT publishes original papers, reviews, and short communications (including viewpoints and perspectives) resulting from research in biotechnology and biotech-associated disciplines. Current Research in Biotechnology is a peer-reviewed gold open access (OA) journal and upon acceptance all articles are permanently and freely available. It is a companion to the highly regarded review journal Current Opinion in Biotechnology (2018 CiteScore 8.450) and is part of the Current Opinion and Research (CO+RE) suite of journals. All CO+RE journals leverage the Current Opinion legacy-of editorial excellence, high-impact, and global reach-to ensure they are a widely read resource that is integral to scientists' workflow.