Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis

Yanting Zhao; Ziheng Yan; Kai Song; Yanbing Li; Leiming Shen; Yiming Cui; Zongmin Du; Ruifu Yang; Yajun Song; Lan Jing; Yong Zhao

doi:10.1371/journal.pntd.0012167

Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis

PLoS Negl Trop Dis. 2024 May 3;18(5):e0012167. doi: 10.1371/journal.pntd.0012167. eCollection 2024 May.

Authors

Yanting Zhao^{1

2}, Ziheng Yan², Kai Song², Yanbing Li^{2

3}, Leiming Shen², Yiming Cui², Zongmin Du^{2

4}, Ruifu Yang^{2

4}, Yajun Song^{2

4}, Lan Jing¹, Yong Zhao^{2

4}

Affiliations

¹ College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.
² State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
³ Department of Laboratory Medicine, Xiangya Hospital of Central South University, Changsha, China.
⁴ Beijing Key Laboratory of POCT for Bioemergency and Clinic, Beijing, China.

Abstract

Background: Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples.

Methods: Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR).

Results: The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103-106 CFU/sample for soil and liver samples.

Conclusion: The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.

Copyright: © 2024 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Publication types

Evaluation Study

MeSH terms

Animals
Bacterial Proteins / genetics
DNA, Bacterial / genetics
Humans
Limit of Detection
Liver / microbiology
Mice
Plague* / diagnosis
Plague* / microbiology
Polymerase Chain Reaction / methods
Reproducibility of Results
Sensitivity and Specificity*
Soil Microbiology*
Yersinia pestis* / genetics
Yersinia pestis* / isolation & purification

Grants and funding

This work was supported by the National Key Research and Development Program (CN) (2022YFC2604200 to YZ) and the State Key Laboratory of Pathogen and Biosecurity (CN) (SKLPBS2112 to YZ). The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.