College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
PLoS Negl Trop Dis. 2024 May 3;18(5):e0012167. doi: 10.1371/journal.pntd.0012167. eCollection 2024 May.
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.
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).
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.
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.
鼠疫由鼠疫耶尔森菌引起，是一种对人类健康构成重大威胁的人畜共患病。核酸检测对于鼠疫监测和快速检测鼠疫耶尔森菌至关重要。然而，土壤和动物组织等复杂样本中的抑制剂常常会阻碍核酸检测，导致低浓度鼠疫耶尔森菌的识别率降低。为了解决这一挑战，我们开发了一种灵敏且特异的检测土壤和动物组织样本中鼠疫耶尔森菌 DNA 的滴液数字聚合酶链反应（ddPCR）检测方法。
使用鼠疫耶尔森菌的三个基因（ypo2088、caf1 和 pla）开发了多靶点 ddPCR 检测方法。评估了细菌基因组 DNA 样本的检测限（LoD）、重现性和特异性。分别评估了该检测方法从模拟土壤和小鼠肝组织样本中检测低浓度鼠疫耶尔森菌 DNA 的能力，并与定量实时 PCR（qPCR）进行了比较。
结果表明，ddPCR 的 LoD 范围为 6.2 至 15.4 个拷贝/反应，对于鼠疫耶尔森菌具有良好的重现性和高度特异性。通过测试 130 份污染有鼠疫耶尔森菌的土壤和小鼠肝组织样本，ddPCR 检测方法的灵敏度优于研究中使用的 qPCR 检测方法，其 LoD 分别为 102 个菌落形成单位（CFU）/100mg 土壤和 103 CFU/20mg 肝。此外，该检测方法在土壤和肝样本中，103-106 CFU/样本的范围内具有良好的 Y. pestis 定量线性（R2=0.99）。
ddPCR 检测方法在检测土壤和小鼠组织样本中的鼠疫耶尔森菌 DNA 方面表现出良好的性能，对于提高鼠疫监测中低浓度鼠疫耶尔森菌的检测率以及促进鼠疫病例的早期诊断具有很大的潜力。
