Detection of pathogenic Leptospira spp. by RPA-NALFIA targeting lipL32 gene

Sirawit  Jirawannaporn; Umaporn  Limothai; Sasipha  Tachaboon; Patcharakorn  Kiatamornrak; Watchadaporn  Chaisuriyong; Nattachai  Srisawat

Authors

Sirawit Jirawannaporn Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand and Faculty of Medicine, Chulalongkorn University, and King Chulalongkorn Memorial Hospital, Bangkok, Thailand
Umaporn Limothai King Chulalongkorn Memorial Hospital, Bangkok, Thailand and Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Sasipha Tachaboon King Chulalongkorn Memorial Hospital, Bangkok, Thailand and Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Patcharakorn Kiatamornrak King Chulalongkorn Memorial Hospital, Bangkok, Thailand and Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Watchadaporn Chaisuriyong King Chulalongkorn Memorial Hospital, Bangkok, Thailand
Nattachai Srisawat Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, King Chulalongkorn Memorial Hospital, Bangkok, Thailand, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand and Faculty of Medicine, Chulalongkorn University, and King Chulalongkorn Memorial Hospital, Bangkok, Thailand

Keywords:

Detection, lateral flow, leptospira spp, leptospirosis

Abstract

Background: Lack of available sensitive point-of-care tests is one of the key challenges limiting the early point-of-care diagnosis of leptospirsis. 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 quantitative polymerase chain reaction (qPCR) and RPA-CRISPR/Cas12a FBDA/LFDA achieved 101 and 102 cell/mL, respectively, and none of the diagnostic tests indicated cross-reactivity with other infectious illnesses. In order to detect leptospirosis in clinical samples, the RPA-NALFIA 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.

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