Abstract
Background: Lack of available sensitive point-of-care tests is one of the key challenges limiting the early point-of-care diagnosis of leptospirosis.Previously, a Recombinase Polymerase Amplification (RPA) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) lipL32 detection platform with high sensitivity and specificity was developed. However, its turnaround time is between one and two hours, and two reactions are required.
Objective: To develop the RPA in combination with a nucleic acid lateral flow immunoassay (NALFIA) detection platform to reduce the turnaround time and make it a one-step reaction.
Methods: RPA combined with nucleic acid lateral flow immunoassay (NALFIA) detection platform was designed to detect the lipL32 gene of pathogenic Leptospira spp.
Results: In pure culture, the limit of detection (LOD) for RPA-NALFIA was 105 cells/mL, whereas qPCR and RPACRISPR/Cas12a FBDA/LFDA achieved 101 and 102 cells/mL, respectively, and none of the diagnostic tests indicated cross-reactivity with other infectious illnesses. In order to detect leptospirosis in clinical samples, the RPANALFIA LOD did not achieve the standard as expected. The further modification of the test to reach acceptable LOD is still needed.
Conclusion: A single-reaction RPA-NALFIA targeting the lipL32 gene was capable of detecting pathogenic Leptospira spp. within an hour without the need for costly laboratory equipment, but improvements are necessary.
DOI
10.56808/2673-060X.5448
Recommended Citation
Srisawat, Nattachai
(2024)
"Detection of pathogenic Leptospira spp. by RPA-NALFIA targeting lipL32 gene,"
Chulalongkorn Medical Journal: Vol. 67:
Iss.
2, Article 10.
DOI: https://doi.org/10.56808/2673-060X.5448
Available at:
https://digital.car.chula.ac.th/clmjournal/vol67/iss2/10