02174nas a2200253 4500000000100000008004100001260001200042653002500054653000800079653000900087653002700096653002400123653003100147653002100178100001200199700001300211700001900224700001400243700001300257245011900270490000700389520151000396022001401906 2026 d c04/202610aMycobacterium leprae10aNGS10aRLEP10ain-house reference DNA10aMolecular diagnosis10anext-generation sequencing10aquantitative PCR1 aGirma S1 aGemeda M1 aWoldesemayat A1 aBobosha K1 aGadisa E00aDevelopment of an in-house control standard for quantitative polymerase chain reaction-based diagnosis of leprosy.0 v573 a

INTRODUCTION:

To develop an in-house reference control DNA standard targeting the RLEP region of Mycobacterium leprae for use in quantitative polymerase chain reaction (qPCR) diagnostics, aiming to reduce dependence on animal models and external sources.

METHOD:

Previously characterized DNA samples from patients with leprosy were used to amplify and sequence the 450-base pair RLEP region. A specific 131-base pair segment within this region was targeted for qPCR assay development. The amplified products were purified; sequenced using Illumina next-generation sequencing technology; and validated through bioinformatics analyses, including Basic Local Alignment Search Tool (BLAST; National Institutes of Health) alignment and copy number assessment.

RESULTS:

The consensus sequence aligned perfectly with the known RLEP region. The BLAST analysis revealed 37 copies of the target sequence throughout the genome, confirming the sequence's utility as a sensitive diagnostic marker. The qPCR assay successfully detected the target in both reference and clinical samples, with melting curve analysis indicating high specificity.

DISCUSSION:

An in-house M leprae RLEP control standard was successfully developed that provides a reliable, ethical, and resource-efficient alternative to traditional animal-derived controls for leprosy diagnosis using qPCR.

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