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Development of a lateral flow recombinase polymerase amplification assay for rapid and visual detection of

Qinglin Ma1,2, Jilong Yao1, Shixin Yuan1

  • 1Institute of Maternity and Child Medical Research, Shenzhen Maternity and Child Healthcare Hospital, Southern Medical University, No.2004 Hongli Road, Shenzhen, 518028, Guangdong, China.

BMC Infectious Diseases
|February 6, 2019
PubMed
Abstract

Insights

A new lateral flow recombinase polymerase amplification (LF-RPA) assay offers rapid and accurate detection of Cryptococcus species DNA in cerebrospinal fluid. This sensitive method aids in the preliminary screening of cryptococcal meningitis.

Area of Science:

  • Medical Diagnostics
  • Molecular Biology
  • Infectious Diseases

Background:

  • Definitive diagnosis of cryptococcal meningitis requires identification of Cryptococcus neoformans and/or C. gattii in cerebrospinal fluid (CSF).
  • Traditional methods like India ink staining and culture have limitations, and cryptococcal antigen detection can be affected by the high-dose hook effect.
  • There is a need for improved, rapid, and simple detection methods for Cryptococcus species in CSF.

Purpose of the Study:

  • To introduce and evaluate a novel, rapid, and simple detection method for Cryptococcus neoformans and C. gattii in CSF.
  • To assess the sensitivity, specificity, and optimal conditions of the developed assay.

Main Methods:

  • Development of a lateral flow strips combined with recombinase polymerase amplification (LF-RPA) assay targeting specific DNA sequences of C. neoformans and C. gattii.
  • Evaluation of detection limit, specificity against other pathogens, optimal reaction time, and temperature.
  • Calculation of diagnostic parameters using 114 clinical CSF specimens and comparison with existing methods.

Main Results:

  • The LF-RPA assay detected as little as 0.64 pg of genomic DNA of C. neoformans within 10 minutes, demonstrating high specificity for Cryptococcus species.
  • The assay performed well across a wide temperature range (25–45°C), and both cell lysates and genomic DNA yielded similar results.
  • Overall sensitivity and specificity were 95.2% and 95.8%, respectively, in clinical specimens.

Conclusions:

  • The developed LF-RPA system is a sensitive and specific method for visible, rapid, and accurate detection of Cryptococcus species in CSF.
  • This assay shows potential for clinical preliminary screening of cryptococcal meningitis, offering a valuable diagnostic tool.
Keywords:
Cerebral spinal fluidCryptococcus gattiiCryptococcus neoformansLateral flow stripsRecombinase polymerase amplificationVisual detection

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