AIMS: Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae's DNA detection based on Polimerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence.
METHODS AND RESULTS: The specific target region, RLEP, of M. leprae's genome was selected based on comparative genomics. After confirming the specificity of this region, using BlastN analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, while the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carry out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, while LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per milliliter, while LP1/LP2 was 1,000 copies of bacterial genomes per milliliter.
CONCLUSIONS: In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy.
SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.
BT - Journal of applied microbiology C1 - https://www.ncbi.nlm.nih.gov/pubmed/31981442 DA - 01/2020 DO - 10.1111/jam.14592 J2 - J. Appl. Microbiol. LA - eng N2 -AIMS: Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae's DNA detection based on Polimerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence.
METHODS AND RESULTS: The specific target region, RLEP, of M. leprae's genome was selected based on comparative genomics. After confirming the specificity of this region, using BlastN analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, while the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carry out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, while LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per milliliter, while LP1/LP2 was 1,000 copies of bacterial genomes per milliliter.
CONCLUSIONS: In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy.
SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.
PY - 2020 T2 - Journal of applied microbiology TI - Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy. SN - 1365-2672 ER -