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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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Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR
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Tick-Borne Pathogens Screening Using a Multiplex Real-Time Polymerase Chain Reaction-Based Method.

Sergio Andres Cardenas-Cadena1, Maria Eugenia Castañeda-Lopez1, Fabiana Esther Mollinedo-Montaño1

  • 1Molecular Medicine Laboratory, Unidad Académica de Medicina Humana y Ciencias de la Salud, Universidad Autónoma de Zacatecas, Zacatecas, 98160, México.

Acta Parasitologica
|August 2, 2023
PubMed
Summary

A new multiplex quantitative real-time polymerase chain reaction (qPCR) assay efficiently detects multiple tick-borne pathogens simultaneously. This cost-effective method aids in timely diagnosis and treatment of tick-borne diseases.

Keywords:
DiagnosticsMultiplex PCRPCR efficiencySYBR GreenTick-borne pathogens

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Area of Science:

  • Veterinary Medicine
  • Molecular Diagnostics
  • Public Health

Background:

  • Tick-borne diseases pose significant risks to human and animal health.
  • Accurate and rapid diagnostics are crucial for effective disease management.
  • Current diagnostic methods can be time-consuming and costly.

Purpose of the Study:

  • To develop a cost-effective, user-friendly multiplex quantitative real-time polymerase chain reaction (qPCR) assay.
  • To detect multiple tick-borne pathogens in a single reaction.
  • To improve the diagnosis of tick-borne diseases.

Main Methods:

  • In silico PCR was used to design primers for Rickettsia spp., Borrelia spp., and Ehrlichia spp.
  • Single and multiplex qPCR assays were standardized.
  • 800 samples were screened for pathogen detection.

Main Results:

  • Multiplex qPCR successfully identified Ehrlichia spp. and Borrelia spp. with a 10-copy limit of detection.
  • Rickettsia spp. were detected with a 100-copy limit of detection.
  • High PCR efficiency (90-100%) and correlation (R²=0.998-0.996) were achieved.

Conclusions:

  • The multiplex qPCR assay enables simultaneous detection of three key tick-borne pathogens.
  • This method offers a significant advantage for molecular diagnostics of tick-borne diseases.
  • The assay facilitates timely intervention, improving patient outcomes and reducing disease complications.