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Volume 13
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10.3390/microorganisms13071596
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Article

Development of a Strain-Specific Detection and Quantification Method for Bifidobacterium animalis subsp. lactis HN019 Using WGS-SNP Analysis and qPCR

by
Da Mao
1,†,
Lei Zhao
2,*,†,
Bo Zhao
1,
Hongbin Xu
2 and
Qinghe Zhang
1
1
Division of Chemical Metrology and Analytical Science, National Institute of Metrology, Beijing 100029, China
2
Key Laboratory of Milk and Dairy Products Detection and Monitoring Technology, State Administration for Market Regulation, Shanghai Institute of Quality Inspection and Technical Research, Shanghai 200233, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2025, 13(7), 1596; https://doi.org/10.3390/microorganisms13071596
Submission received: 15 April 2025 / Revised: 24 June 2025 / Accepted: 3 July 2025 / Published: 7 July 2025
(This article belongs to the Special Issue Microbial Safety and Beneficial Microorganisms in Foods)
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Abstract

Accurate quantification of Bifidobacterium animalis subsp. lactis HN019, a clinically validated probiotic strain conferring immune modulation, gastrointestinal health, and gut barrier integrity benefits, is essential for diverse applications. To address the critical need for strain-specific detection, we developed a quantitative PCR (qPCR) assay targeting a unique single-nucleotide polymorphism (SNP) within the galK gene, identified through comparative whole-genome sequencing (WGS) analysis of 31 B. animalis subsp. lactis strains. The assay exhibited exceptional specificity, distinguishing HN019 from 19 other Bifidobacterium strains. Sensitivity tests indicated a detection limit of 0.5 pg of DNA and 103 CFU/mL of bacterial cells, making it suitable for industrial-scale applications. Additionally, the method exhibited strong repeatability, reproducibility across different qPCR platforms, and resistance to interference from high cell density of B. animalis subsp. lactis DSMZ 10140. Successful quantification of HN019 in complex multi-strain probiotic powders confirmed its practical reliability. This work establishes a rapid, robust, and scalable tool for precise probiotic strain tracking, addressing critical quality control and regulatory compliance needs within the rapidly expanding probiotic industry.
Keywords: Bifidobacterium animalis subsp. lactis; strain-specific; identification; quantification; WGS-SNP Bifidobacterium animalis subsp. lactis; strain-specific; identification; quantification; WGS-SNP

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MDPI and ACS Style

Mao, D.; Zhao, L.; Zhao, B.; Xu, H.; Zhang, Q. Development of a Strain-Specific Detection and Quantification Method for Bifidobacterium animalis subsp. lactis HN019 Using WGS-SNP Analysis and qPCR. Microorganisms 2025, 13, 1596. https://doi.org/10.3390/microorganisms13071596

AMA Style

Mao D, Zhao L, Zhao B, Xu H, Zhang Q. Development of a Strain-Specific Detection and Quantification Method for Bifidobacterium animalis subsp. lactis HN019 Using WGS-SNP Analysis and qPCR. Microorganisms. 2025; 13(7):1596. https://doi.org/10.3390/microorganisms13071596

Chicago/Turabian Style

Mao, Da, Lei Zhao, Bo Zhao, Hongbin Xu, and Qinghe Zhang. 2025. "Development of a Strain-Specific Detection and Quantification Method for Bifidobacterium animalis subsp. lactis HN019 Using WGS-SNP Analysis and qPCR" Microorganisms 13, no. 7: 1596. https://doi.org/10.3390/microorganisms13071596

APA Style

Mao, D., Zhao, L., Zhao, B., Xu, H., & Zhang, Q. (2025). Development of a Strain-Specific Detection and Quantification Method for Bifidobacterium animalis subsp. lactis HN019 Using WGS-SNP Analysis and qPCR. Microorganisms, 13(7), 1596. https://doi.org/10.3390/microorganisms13071596

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