{"title":"qPCR detection of <i>Mycobacterium leprae</i> DNA in urine samples of leprosy patients using the <i>Rlep</i> gene target.","authors":"D Diana, M C Harish","doi":"10.3389/fmolb.2024.1435679","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Leprosy, a chronic infectious disease caused by <i>Mycobacterium leprae</i>, continues to pose a public health challenge in many parts of the world. Early and accurate diagnosis is crucial for effective treatment and prevention of disabilities associated with the disease. Molecular techniques such as PCR have demonstrated great potential as a diagnostic tool for directly detecting <i>M. leprae</i> DNA in different clinical samples, providing better sensitivity and specificity than conventional diagnostic techniques. The objective of this study was to measure the amount of <i>M. leprae</i> DNA in leprosy patients' urine samples using the <i>Rlep</i> gene target through qPCR.</p><p><strong>Methods: </strong>Different clinical samples such as smear, blood, and urine samples were collected from leprosy patients and healthy individuals. Leprosy patients were classified by the Ridley-Jopling classification. The Ziehl-Neelsen staining method was used for the slit skin smear (SSS) samples, and the bacteriological index (BI) was calculated for leprosy patients. DNA extraction and qPCR were performed for all three types of clinical samples using the <i>Rlep</i> gene target.</p><p><strong>Results: </strong>The <i>Mycobacterial leprae</i> DNA was successfully detected and quantified in all clinical samples across all types of leprosy among all the study groups using the <i>Rlep</i> gene (129 bp) target. The <i>Rlep</i> gene target was able to detect the presence of <i>M. leprae</i> DNA in 100% of urine, 96.1% of blood, and 92.2% of SSS samples of leprosy patients. Urine samples showed significant differences (p < 0.001) between the control and the different clinical forms and between borderline tuberculoid (BT) and pure neuritic leprosy (PNL) cases. There are significant differences in cycle threshold (Ct) values between control cases and clinical categories (p < 0.001), as well as specific differences within clinical categories (p < 0.001), reflecting the variability in bacterial load and detection sensitivity across different sample types and clinical manifestations of leprosy.</p><p><strong>Conclusion: </strong>Overall, this study's findings suggest that the qPCR technique can be used to detect <i>M. leprae</i> DNA in urine samples of leprosy patients using the <i>Rlep</i> gene target. It can also be used for diagnosing the disease and monitoring the effectiveness of anti-leprosy drugs, including multi-drug therapy (MDT), across various leprosy disease groups.</p>","PeriodicalId":12465,"journal":{"name":"Frontiers in Molecular Biosciences","volume":"11 ","pages":"1435679"},"PeriodicalIF":3.9000,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11347395/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Molecular Biosciences","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.3389/fmolb.2024.1435679","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Background: Leprosy, a chronic infectious disease caused by Mycobacterium leprae, continues to pose a public health challenge in many parts of the world. Early and accurate diagnosis is crucial for effective treatment and prevention of disabilities associated with the disease. Molecular techniques such as PCR have demonstrated great potential as a diagnostic tool for directly detecting M. leprae DNA in different clinical samples, providing better sensitivity and specificity than conventional diagnostic techniques. The objective of this study was to measure the amount of M. leprae DNA in leprosy patients' urine samples using the Rlep gene target through qPCR.
Methods: Different clinical samples such as smear, blood, and urine samples were collected from leprosy patients and healthy individuals. Leprosy patients were classified by the Ridley-Jopling classification. The Ziehl-Neelsen staining method was used for the slit skin smear (SSS) samples, and the bacteriological index (BI) was calculated for leprosy patients. DNA extraction and qPCR were performed for all three types of clinical samples using the Rlep gene target.
Results: The Mycobacterial leprae DNA was successfully detected and quantified in all clinical samples across all types of leprosy among all the study groups using the Rlep gene (129 bp) target. The Rlep gene target was able to detect the presence of M. leprae DNA in 100% of urine, 96.1% of blood, and 92.2% of SSS samples of leprosy patients. Urine samples showed significant differences (p < 0.001) between the control and the different clinical forms and between borderline tuberculoid (BT) and pure neuritic leprosy (PNL) cases. There are significant differences in cycle threshold (Ct) values between control cases and clinical categories (p < 0.001), as well as specific differences within clinical categories (p < 0.001), reflecting the variability in bacterial load and detection sensitivity across different sample types and clinical manifestations of leprosy.
Conclusion: Overall, this study's findings suggest that the qPCR technique can be used to detect M. leprae DNA in urine samples of leprosy patients using the Rlep gene target. It can also be used for diagnosing the disease and monitoring the effectiveness of anti-leprosy drugs, including multi-drug therapy (MDT), across various leprosy disease groups.
期刊介绍：
Much of contemporary investigation in the life sciences is devoted to the molecular-scale understanding of the relationships between genes and the environment — in particular, dynamic alterations in the levels, modifications, and interactions of cellular effectors, including proteins. Frontiers in Molecular Biosciences offers an international publication platform for basic as well as applied research; we encourage contributions spanning both established and emerging areas of biology. To this end, the journal draws from empirical disciplines such as structural biology, enzymology, biochemistry, and biophysics, capitalizing as well on the technological advancements that have enabled metabolomics and proteomics measurements in massively parallel throughput, and the development of robust and innovative computational biology strategies. We also recognize influences from medicine and technology, welcoming studies in molecular genetics, molecular diagnostics and therapeutics, and nanotechnology.
Our ultimate objective is the comprehensive illustration of the molecular mechanisms regulating proteins, nucleic acids, carbohydrates, lipids, and small metabolites in organisms across all branches of life.
In addition to interesting new findings, techniques, and applications, Frontiers in Molecular Biosciences will consider new testable hypotheses to inspire different perspectives and stimulate scientific dialogue. The integration of in silico, in vitro, and in vivo approaches will benefit endeavors across all domains of the life sciences.
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