Evaluation of BOX-PCR and ERIC-PCR as Molecular Typing Tools for PathogenicLeptospira

Abstract

In the last decades, leptospirosis had gained public health concern due to morbidity and mortality rates caused by pathogenic Leptospira. The need for rapid and robust molecular typing methods to differentiate this zoonotic pathogen is of utmost importance. Various studies had been conducted to determine the genetic relatedness of Leptospira isolates using molecular typing methods. In this study, 29 pathogenic Leptospira isolates from rat, soil, and water samples in Sarawak, Malaysia, were characterized using BOX-PCR and ERIC-PCR. The effectiveness of these two methods with regard to the ease of interpretation, reproducibility, typeability, and discriminatory power was also being evaluated. Using BOX-PCR, six clusters and 3 single isolates were defined at a genetic distance percentage of 11.2%. ERIC-PCR clustered the isolates into 6 clusters and 2 single isolates at a genetic distance percentage of 6.8%. Both BOX-PCR and ERIC-PCR produced comparable results though the discriminatory index for ERIC-PCR (0.826) was higher than that for BOX-PCR (0.809). From the constructed dendrogram, it could be summarized that the isolates in this study were highly heterogeneous and genetically diverse. The findings from this study indicated that there is no genetic relatedness among the pathogenic Leptospira isolates in relation to the locality, source, and identity, with some exceptions. Out of the 29 pathogenic Leptospira isolates studied, BOX-PCR and ERIC-PCR successfully discriminated 4 isolates (2 isolates each) into the same cluster in relation to sample sources, as well as 2 isolates into the same cluster in association with the sample locality. Future studies shall incorporate the use of other molecular typing methods to make a more thorough comparison on the genetic relatedness of pathogenic Leptospira.

Molecular Characterization of Carbapenemase-Producing Klebsiella pneumoniae Isolated from Egyptian Pediatric Cancer Patients Including a Strain with a Rare Gene-Combination of β-Lactamases

“…The PCR reaction contained 12.5 µL of DreamTaq Green PCR Master Mix (2X), 50 ng of bacterial DNA template, 2 µL from the ERIC primers ( Table 1 ), and nuclease-free water to complete the total volume to 25 µL. According to the protocol described by Bilung and colleagues 24 with slight modifications, the conditions used were as follows: initial denaturation for 15 min at 95°C followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 45°C, the extension for 8 min at 72°C, and a final extension for 15 min at 72°C.…”

Outbreak of Ralstonia mannitolilytica bacteraemia in patients undergoing haemodialysis at a tertiary hospital in Pretoria, South Africa

“…Each 25 μl reaction mixture contained HotStar Taq DNA polymerase, 10× primer mix, RNase free water and template DNA, using 2 μL template DNA (< 1 μg DNA/25 μL). The PCR amplification conditions were; initial denaturation at 95 °C for 15 s, denaturation at 95 °C for 10 s for 45 cycles, annealing at 65 °C for 10 s, extension at 72 °C for 20 s and final extension at 72 °C for 5 min [ 7 ]. A negative control (no template) and positive control ( Pseudomonas aeruginosa ATCC 27853) were included in all PCR assays.…”

Molecular determination of genetic diversity among Campylobacter jejuni and Campylobacter coli isolated from milk, water, and meat samples using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR)

“…The capacity of a technique to give the same result when repeated tests are carried out on the same isolate is recognized as reproducibility [ 22 ]. The ERIC-PCR analysis of the DNA fingerprints result can be directly correlated within a single PCR experiment.…”

“…This technique is a strong tool for the exploration of prokaryotic genomes and has been reported to have improved reproducibility and high discriminatory power [ 21 ]. ERIC-PCR is a repetitive element-based PCR technique [ 22 ]. The pros of ERIC-PCR over other molecular typing techniques include the capacity to distinguish between closely related bacteria strains as well as being quick, simple, cheap, dependable, and high-throughput genotyping method [ 23 ].…”