Laboratory medicine · 2026
Development of an in-house control standard for quantitative polymerase chain reaction-based diagnosis of leprosy.
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Girma Selfu, Gemeda Mesfin Tafesse, Woldesemayat Adugna Abdi, Bobosha Kidist, Gadisa Endalamaw
Аннотация
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.
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.