Development of a high-copy target enhanced multiplexed crRNA-based, amplification-free detection assay (HiTECT) for Brucella spp.

BACKGROUND

Brucellosis, a zoonotic disease caused by Brucella species, continues to pose major health challenges for humans and livestock, along with significant economic losses. Traditional serological tests are widely used but are limited, as they cannot reliably distinguish between active infection and past exposure. Molecular tools such as PCR and real-time PCR offer high sensitivity and specificity; however, their dependence on sophisticated equipment and trained personnel restricts their use outside well-equipped laboratories. More recently, isothermal techniques such as LAMP and RPA have emerged as faster, simpler alternatives, and their integration with CRISPR-based platforms has further enhanced detection capabilities. Nevertheless, these methods still face challenges, including the requirement for pre-amplification and the risk of contamination, which limit their practical application in field conditions. METHODS AND

RESULTS

In this study, we developed an amplification-free CRISPR-Cas12a assay—HiTECT (High-copy Target Enhanced CRISPR Test)—targeting the high-copy IS711 insertion element for thermocycler free rapid detection of Brucella spp. Using a multiplexed crRNA strategy, HiTECT enabled robust visual detection without nucleic acid amplification. Under optimal conditions (100 nM LbCas12a with a 1:1 Cas12a–crRNA ratio) and employing three crRNAs, fluorescence was significantly enhanced. The assay demonstrated an analytical sensitivity of 461.71 ag/µL of genomic DNA (0.92 fg/ reaction) and approximately 6*10⁴ CFU/mL for Brucella suis 1330. HiTECT was found very specific as it could detect all 16 field isolates and four reference Brucella strains, with no cross-reactivity to any of the non Brucella bacterial species, and showed substantial concordance with real-time PCR (κ = 0.65).

CONCLUSION

HiTECT provides a robust, amplification-free, field-deployable platform for rapid and sensitive detection of Brucella spp., offering clear advantages for resource-limited settings.