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Journal of clinical microbiology2023; 61(3); e0137522; doi: 10.1128/jcm.01375-22

Development and Application of Real-Time PCR Assay for Detection of Salmonella Abortusequi.

Abstract: Salmonella enterica subsp. serovar Abortusequi is a major pathogen in horse and donkey herds, causing abortion in pregnant equids and resulting in enormous economic losses. A rapid and reliable method is urgently needed to detect . Abortusequi in herds where the disease is suspected. To achieve this goal, a TaqMan-based real-time PCR assay targeting the gene for the flagellin protein phase 2 antigen FljB was developed. This real-time PCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the assay was 30 copies/μL of standard plasmid and 10 CFU/μL of bacterial DNA. Furthermore, 540 clinical samples, including 162 tissue, 192 plasma, and 186 vaginal swab samples collected between 2018 and 2021 in China, were tested to assess the performance of the developed assay. Compared to the gold standard method of bacterial isolation, the real-time PCR assay exhibited 100% positive agreement for all tissue, plasma and vaginal swab tests. Additionally, this assay detected DNA from Abortusequi from 56.7% (34/60) culture-negative tissue and 22.9% (41/179) culture-negative vaginal swab samples from infected equids. Receiver operating characteristic analysis demonstrated that the results of the developed real-time PCR assays were in significant agreement with those of the culture method. The real-time PCR assay can be completed within 45 min of extraction of DNA from samples. Our results show that this assay could serve as a reliable tool for the rapid detection of Abortusequi in tissue, plasma, and vaginal swab clinical samples.
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Publication Date: 2023-03-01 PubMed ID: 36856425PubMed Central: PMC10035326DOI: 10.1128/jcm.01375-22Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research is about the development of a rapid and reliable method, known as a Taqman-based real-time PCR assay, for detecting Salmonella enterica, a major bacterial pathogen in horse and donkey herds, that frequently results in pregnancy loss and consequent economic losses.

Understanding the Problem

  • The study’s primary purpose was to address the issue of Salmonella enterica subsp. serovar Abortusequi, a serious bacterial infection in horse and donkey herds that often leads to abortion in pregnant animals, resulting in significant economic losses.
  • The detection of this bacteria has been challenging due to the lack of a rapid and reliable method. Hence, there was an urgent need to develop an efficient testing method that was both accurate and quick.

Method Developed

  • The authors developed a TaqMan-based real-time PCR (Polymerase Chain Reaction) assay for this purpose. This sophisticated technology targets the gene responsible for the second phase of the flagellin protein antigen, FljB, found in the bacteria.
  • The real-time PCR assay demonstrated high specificity, sensitivity, and reproducibility, meaning it was reliable in detecting the bacteria accurately.
  • The detection limit of the assay was notably low, capturing 30 copies/μL of standard plasmid and 10 CFU/μL of bacterial DNA. This high sensitivity is significant as it means the test can identify even small amounts of the bacteria.

Performance Assessment of the Assay

  • The researchers tested 540 clinical samples that included tissue, plasma, and vaginal swab samples, collected between 2018 and 2021 in China.
  • The results of the real-time PCR assay were compared to the gold standard method – bacterial isolation. In all tissue, plasma, and vaginal swab tests, the PCR assay displayed 100% positive agreement, establishing the reliability of the test.
  • The test could also detect bacterial DNA in 56.7% of culture-negative tissue and 22.9% of culture-negative vaginal swab samples, from infected equids.
  • The Receiver operating characteristic analysis, a statistical technique used to evaluate the performance of diagnostic tests, indicated that the results of the real-time PCR assays were significantly in agreement with those of the culture method.

Speed and Application

  • The real-time PCR assay can be completed within 45 minutes of extracting DNA from samples, demonstrating the test’s efficiency and speed.
  • The researchers concluded that this assay could serve as a reliable tool for rapidly detecting Salmonella enterica subsp. serovar Abortusequi in tissue, plasma, and vaginal swab clinical samples from horses and donkeys.

Cite This Article

APA
Wang J, Guo K, Li S, Liu D, Chu X, Wang Y, Guo W, Du C, Wang X, Hu Z. (2023). Development and Application of Real-Time PCR Assay for Detection of Salmonella Abortusequi. J Clin Microbiol, 61(3), e0137522. https://doi.org/10.1128/jcm.01375-22

Publication

Journal of clinical microbiology
ISSN: 1098-660X
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 61
Issue: 3
Pages: e0137522
PII: e01375-22

Researcher Affiliations

Wang, Jinhui
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Guo, Kui
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Li, Shuaijie
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Liu, Diqiu
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Chu, Xiaoyu
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Wang, Yaoxin
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Guo, Wei
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Du, Cheng
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Wang, Xiaojun
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Hu, Zhe
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.

MeSH Terms

  • Pregnancy
  • Female
  • Animals
  • Horses / genetics
  • Salmonella enterica / genetics
  • Real-Time Polymerase Chain Reaction
  • Reproducibility of Results
  • Salmonella Infections, Animal / diagnosis
  • Salmonella Infections, Animal / microbiology
  • Salmonella / genetics
  • DNA, Bacterial / genetics
  • Sensitivity and Specificity

Conflict of Interest Statement

The authors declare no conflict of interest.

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Citations

This article has been cited 10 times.
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