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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Molecular Investigation of Small Ruminant Abortions Using a 10-Plex HRM-qPCR Technique: A Novel Approach in Routine

Ioannis Gouvias1, Marios Lysitsas1, Apostolos Batsidis2

  • 1Faculty of Veterinary Science, University of Thessaly, 43100 Karditsa, Greece.

Microorganisms
|August 29, 2024
PubMed
Summary

A new multiplex High-Resolution Melting (HRM) analysis coupled with qPCR accurately detects 10 ovine and caprine abortogenic pathogens. This cost-effective method identified high prevalence of Coxiella burnetii and Chlamydophila spp., revealing previously unreported pathogens in Greece.

Keywords:
Anaplasma phagocytophilumBrucella spp.Campylobacter fetusChlamydophila spp.Coxiella burnetiiGreeceHRMNeospora caninumSalmonella spp.Toxoplasma gondiiabortogenic pathogensqPCRsmall ruminants

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Area of Science:

  • Veterinary Microbiology
  • Molecular Diagnostics
  • Ruminant Health

Background:

  • Abortion in sheep and goats poses significant economic and reproductive challenges in livestock.
  • Accurate and rapid diagnosis of abortogenic pathogens is crucial for effective disease management.
  • Existing diagnostic methods may not cover the broad spectrum of potential causative agents.

Purpose of the Study:

  • To evaluate a novel High-Resolution Melting (HRM) analysis technique combined with qPCR for simultaneous detection of 10 ruminant abortogenic pathogens.
  • To investigate the prevalence of these pathogens in ovine and caprine abortions in Greece.
  • To assess the diagnostic performance of the multiplex HRM-qPCR assay.

Main Methods:

  • A commercially available multiplex HRM-qPCR kit (ID Gene™ Ruminant Abortion Multiplex HRM) was used.
  • 264 vaginal swabs from aborted sheep and goats in Greece were analyzed.
  • Results were compared with a well-established commercial qPCR kit for key pathogens.

Main Results:

  • High prevalence of Coxiella burnetii (48.9%) and Chlamydophila spp. (42.4%) was observed.
  • Near-perfect agreement was found between the HRM-qPCR assay and a standard qPCR kit.
  • Campylobacter fetus and Neospora caninum were identified for the first time in small ruminants in Greece.
  • Mixed infections were detected in 35.6% of cases.

Conclusions:

  • The multiplex HRM-qPCR assay is an accurate, cost-effective, and efficient tool for diagnosing ovine and caprine abortions.
  • The study highlights the significant role of C. burnetii and Chlamydophila spp., and suggests an underestimation of other pathogens.
  • The findings underscore the importance of comprehensive etiological investigations in ruminant abortions, including previously unreported agents.