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

Yanting Zhao1,2, Ziheng Yan2, Kai Song2

  • 1College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.

Plos Neglected Tropical Diseases
|May 3, 2024
PubMed
Abstract

Insights

A new droplet digital PCR (ddPCR) assay effectively detects Yersinia pestis DNA in soil and animal tissues. This sensitive method improves plague surveillance by identifying low bacterial concentrations, aiding early diagnosis.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Infectious Diseases

Background:

  • Plague, caused by Yersinia pestis, is a significant zoonotic threat.
  • Nucleic acid tests are vital for Y. pestis detection but hindered by inhibitors in environmental and tissue samples.
  • Developing sensitive detection methods for Y. pestis in complex matrices is crucial for surveillance.

Purpose of the Study:

  • To develop and validate a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for Yersinia pestis DNA detection.
  • To evaluate the assay's performance in complex samples like soil and animal tissues.
  • To compare the ddPCR assay's sensitivity against quantitative real-time PCR (qPCR).

Main Methods:

  • A multi-target ddPCR assay was designed targeting three Y. pestis genes (ypo2088, caf1, and pla).
  • Assay performance was evaluated for limits of detection (LoD), reproducibility, and specificity using bacterial genomic DNA.
  • The ddPCR assay's ability to detect low Y. pestis concentrations in spiked soil and mouse liver samples was assessed and compared to qPCR.

Main Results:

  • The ddPCR assay demonstrated low LoDs (6.2–15.4 copies/reaction) with high specificity and reproducibility.
  • Compared to qPCR, ddPCR showed superior sensitivity in detecting Y. pestis in spiked soil (LoD: 102 CFU/100 mg) and liver (LoD: 103 CFU/20 mg) samples.
  • The assay exhibited excellent quantitative linearity (R2 = 0.99) for Y. pestis concentrations ranging from 10^3 to 10^6 CFU/sample.

Conclusions:

  • The developed ddPCR assay is highly effective for detecting Yersinia pestis DNA in soil and animal tissues.
  • This assay holds significant potential for enhancing plague surveillance by improving the detection rate of low bacterial loads.
  • The ddPCR method can facilitate earlier diagnosis of plague cases, contributing to public health efforts.

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