Rapid differentiation of Mycobacterium tuberculosis and M . bovis by high 1 resolution melt curve analysis 2 3 4

45 46 Identification and characterization of the Mycobacterium tuberculosis strains is important for 47 clinical and therapeutic management of tuberculosis. Real time PCR with high-resolution melt 48 assay were found to improve the diagnostic process. The assay includes differentiation between 49 M. tuberculosis and M. bovis based on one SNP in the narGHJI and oxyR genes, and 50 determination of M. bovis based on the RD1 region. This assay correctly identified the tested 51 seven Mycobacterium reference strains and fifty two clinical samples with a sensitivity of 2 pg 52 DNA. This will help in prescribing adequate treatment and monitoring disease dynamics. 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 on S etem er 3, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom Mycobacterium tuberculosis complex (MTC) includes M. tuberculosis, M. africanum both of 75 which are considered human pathogens while M. microti and M. bovis usually are associated 76 with animal infections. Though M. tuberculosis (MTB) is the main cause of human tuberculosis 77 (TB), it has been estimated that M. bovis responsible for 10 to 15% of new human TB in the 78 developing countries (1). The attenuated tuberculosis vaccine strain, Bacilli Calette Guerin 79 (BCG), can also cause human TB especially in cases diagnosed with cellular immunodeficiency 80 (2) or among neonates and children in endemic regions that were vaccinated (1, 3). In 2003, a 81 Palestinian study described an outbreak of BCG complications in the Gaza strip of the 82 Palestinian territories. It affected 225 infants (average age was 4 months) with a complication 83 rate of 36.61 per 1000 vaccinations (4). Thus, differentiation and identification of the MTB from 84 other members of the MTC should improve the clinical and the therapeutic management of TB. 85 Moreover, it contributes to the understanding of the TB epidemiology. Several molecular 86 methods have been reported for genotyping these two pathogens, e.g., Multiplex PCR, PCR 87 restriction analysis, alleles specific PCR and real-time PCR using FRET probes (5, 6, 7, 8, 9). 88 However, these methods are time consuming, expensive and complicated. Comparative genome 89 analysis has shown that M. bovis have numerous single nucleotide polymorphisms (SNPs) 90 compared to MTB (10). Of these SNPs, the C/T transition at position -215 upstream of the GTG 91 start codon in the promoter region of the narGHJI gene was hypothesized to be responsible for 92 the differential Nitrate Reductase activity between MTB and M. bovis (11). Another 93 polymorphic nucleotide was identified in the oxyR gene (6, 12) and was found to be specific for 94 M. bovis. In this study, we describe the use of high resolution melt curve analysis (HRM) to 95 differentiate between M. bovis and M. tuberculosis by two stepwise reactions. The first reaction 96 is based on the T/C transition at position -215 in the promoter region of the narGHJI gene which 97 differentiates between MTB and all other members of the MTC (M. africanum, M. microti and 98 M. bovis) (6). The second reaction is based on the A/G polymorphism in the oxyR gene which is 99 on S etem er 3, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom specific to M. bovis and therefore, differentiates between M. bovis and M. africanum or M. 100 microti. Such definitive differentiation is essential, even if they are confined to different 101 restricted geographical areas and to different host species. In addition, all M. bovis strains were 102 further identified targeting the region of differences 1 (RD1) a 9,650 bp deletion, which appears 103 as a specific marker for M. bovis BCG) using the HRM assay. The present study was aimed at 104 developing a rapid assay for differentiation between M. bovis and M. tuberculosis with minimal 105 requirements of cost and time. 106 The study included 52 samples: seven were previously identified as M. tuberculosis (13) and 107 kindly provided from the Austrian Agency for Health and food safety, Vienna, Austria, fifteen 108 clinical isolates recovered from sputum samples at the central laboratories of the Palestinian 109 Ministry of Health and 30 DNA samples extracted from Ziehl-Neelsen stained sputum smears ( 110 during the years 2005-2009). All archival samples and isolates had previously identified by 111 IS6110-based PCR to the complex level. Purified DNA from the reference strains M. 112 tuberculosis (H37Rv), M. bovis, M. bovis BCG and from non-tuberculosis mycobacteria (NTM) 113 (M. phlei, M. avium, M. intracellulare and M. kansasii) were generously provided from The 114 Hebrew University-Hadassah Medical School, Jerusalem, Israel. 115 Ten samples from TB negative sputum smears (confirmed negatives by IS6110based PCR) 116 were included as negative controls in the study. 117 The Ziehl-Neelsen stained material was scraped off from the microscopic slides after addition of 118 200 μl of tissue lysis buffer, and then processed by proteinase k digestion, followed by extraction 119 according to manufactures instruction (High pure PCR template preparation, Mannheim, 120