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Despite the high sensitivity and specificity of PCR for infectious disease diagnostics, it has presented low sensitivity for Mycobacterium leprae DNA detection in the tuberculoid pole (TT and BT) of leprosy. In order to demonstrate the effect of amplicon size on the efficacy of PCR detection of M. leprae DNA in skin lesions of leprosy patients, two pairs of primers targeting the M. leprae genomic DNA, RLEP3 (X17153), were used to amplify fragments of 372 and 130-bp until their PCR end-points were reached after 40 reaction cycles. Skin biopsies of leprosy lesions in 110 non-treated patients were used for bacilloscopy index (BI) analysis and PCR tests. The 130-bp fragment was detected in 73.6% of samples (81/110), and classified as TT (40%), BT (55.5%), and 100% of BB, BL and LL. The 372-bp fragment was detected in 52.7% and classified as TT (13.3%), BT (33.3%), BB (64.7%), BL (83.3%), and LL (95.2%). The BI of biopsies was positive in 39.1% of samples, classified as TT (0%), BT (2.2%), BB (64.7%), BL (91.6%), and LL (95.2%). The shorter amplicon (130-bp) has improved diagnosis by 20.9 and 34.5% in relation to the 372-bp fragment and the BI, respectively, and has shown a superior sensitivity (73.6%), specificity (100%) and accuracy (86.2%). The 130-bp amplicon could not detect % of positive BI of biopsies in BT cases. Therefore, for confirmatory diagnosis, we propose the use of PCR detection of the 130-bp genomic target, especially when the tuberculoid pole forms are considered, which has reached 51.6% of positivity in this group.