Open Access
Research (Published online: 17-06-2023)
24. Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii
Vena Chupia, Jirapat Ninsuwon, Montira Intanon, and Surachai Pikulkaew
Veterinary World, 16(6): 1356-1362

Vena Chupia: Department of Veterinary Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, 50100, Thailand; Research Center of Producing and Development of Products and Innovations for Animal Health and Production, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, 50100, Thailand.
Jirapat Ninsuwon: Veterinary Diagnostic Center, Chiang Mai University Animal Hospital, Faculty of Veterinary Medicine, Chiang Mai University, 50100, Thailand.
Montira Intanon: Department of Veterinary Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, 50100, Thailand; Research Center of Producing and Development of Products and Innovations for Animal Health and Production, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, 50100, Thailand.
Surachai Pikulkaew: Research Center of Producing and Development of Products and Innovations for Animal Health and Production, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, 50100, Thailand; Department of Food Animal Clinics, Faculty of Veterinary Medicine, Chiang Mai University, 50100, Thailand.

doi: 10.14202/vetworld.2023.1356-1362

Article history: Received: 22-02-2023, Accepted: 23-05-2023, Published online: 17-06-2023

Corresponding author: Surachai Pikulkaew

E-mail: surapikulkaew@gmail.com

Citation: Chupia V, Ninsuwon J, Intanon M, and Pikulkaew S (2023) Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii, Veterinary World, 16(6): 1356-1362.
Abstract

Background and Aim: Sporothrix schenckii is the causative agent of sporotrichosis, which most commonly causes lymphocutaneous infections in immunocompromised hosts. This pathogen infects dogs, cats, cattle, and buffaloes and can potentially infect humans. Diagnosis by fungal culture is lengthy, and although there are several clinical diagnoses and molecular methods, these are complicated and time-consuming for veterinarians. This study aimed to develop a visual diagnostic assay that is less time-consuming and can be used by veterinarians to screen for sporotrichosis.

Materials and Methods: To develop a loop-mediated isothermal amplification (LAMP) assay for sporotrichosis, primers specific for fragments of the 18S rRNA gene of S. schenckii were designed. Then, the time and temperature were optimized to successfully achieve LAMP. Ten-fold serial dilutions of DNA were used to determine the detection limit using both LAMP and nested polymerase chain reaction (nPCR) assays.

Results: The optimal LAMP conditions were incubation at 73°C for 30 min. Agarose gel electrophoresis revealed a ladder-like pattern of the LAMP product, and a sky-blue color indicated a positive result. A comparison of the LAMP assay with nPCR revealed that it was 10 times more sensitive than nPCR, with a detection limit of 10 pg. The use of a heat box compared with a thermocycler gave the same results.

Conclusion: Loop-mediated isothermal amplification gives good results and may represent a future alternative diagnostic tool for screening fungal pathogens before the results of conventional fungal cultures are received. However, this method should be further studied to clarify its use with clinical samples.

Keywords: loop-mediated isothermal amplification, molecular detection, rapid detection, Sporothrix schenckii, sporotrichosis, visual detection.