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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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Use of FTA Cards for DNA Preservation and Dual-LAMP Analysis of Plasmodium knowlesi and P. cynomolgi.

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Development of a chemical precipitated-LAMP assay for the diagnosis of knowlesi malaria.

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Recombinase-Aided Loop-Mediated Isothermal Amplification on Human Plasmodium knowlesi.

Meng Yee Lai1, Mohd Hafizi Abdul Hamid2, Jenarun Jelip2

  • 1Department of Parasitology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia.

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|February 27, 2024
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Recombinase-aided loop-mediated isothermal amplification (LAMP) rapidly detects Plasmodium knowlesi, the cause of knowlesi malaria. This sensitive, colorimetric assay is ideal for remote diagnostics.

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Area of Science:

  • Molecular Biology
  • Parasitology
  • Medical Diagnostics

Background:

  • Loop-mediated isothermal amplification (LAMP) is a rapid nucleic acid amplification method.
  • Conventional LAMP requires specific temperature control and can be time-consuming.
  • Accurate detection of Plasmodium knowlesi is crucial for malaria diagnosis and treatment.

Purpose of the Study:

  • To develop and evaluate a recombinase-aided LAMP assay for specific amplification of Plasmodium knowlesi 18S sRNA.
  • To assess the sensitivity, specificity, and speed of the developed assay.
  • To establish a colorimetric detection method for Plasmodium knowlesi suitable for resource-limited settings.

Main Methods:

  • Incorporation of a recombinase enzyme into a conventional LAMP assay.
  • Amplification of Plasmodium knowlesi 18S sRNA at a constant temperature (65°C).
  • Colorimetric detection using malachite green dye for visual result interpretation.

Main Results:

  • The recombinase-aided LAMP assay detected Plasmodium knowlesi within 28 minutes.
  • The assay demonstrated high specificity, with no cross-reactivity observed in non-P. knowlesi samples.
  • A detection limit of one copy of P. knowlesi DNA was achieved.
  • Visual color change (green to colorless) allowed for naked-eye detection.

Conclusions:

  • Recombinase-aided LAMP is a sensitive, rapid, and specific diagnostic tool for Plasmodium knowlesi.
  • The colorimetric detection platform makes the assay accessible for use in remote healthcare settings.
  • This method offers a convenient alternative to traditional diagnostic techniques for knowlesi malaria.