Detection of Mycobacterium ulcerans by real-time PCR with improved primers
© The Author(s) 2016
Received: 25 May 2016
Accepted: 1 August 2016
Published: 19 August 2016
Buruli ulcer is a severe skin disease caused by Mycobacterium ulcerans. Real-time PCR targeting the IS2404 sequence has been used as a reliable and rapid method for the diagnosis of Buruli ulcer and detection of M. ulcerans in the environment. The genome of M. ulcerans contains hundreds of IS2404 copies, which have variability in certain sequences. Therefore, the design of new primers specific to conserved IS2404 regions may potentially improve the sensitivity of M. ulcerans detection and, consequently, the diagnosis of Buruli ulcer, thus ensuring timely treatment of the disease.
In silico analysis indicates that DNA sequences of the IS2404 elements are highly variable within a single strain. As the binding sites of conventional IS2404-specific primers used for M. ulcerans detection contain polymorphic sequences, we designed new primers, which enabled the detection of M. ulcerans by real-time PCR with higher sensitivity and similar specificity with respect to that of conventional primers. However, the increase in sensitivity with the new primers depended on the M. ulcerans strain.
The results suggest that real-time PCR based on the new primers could improve Buruli ulcer diagnosis and M. ulcerans detection in environmental samples.
Buruli ulcer, a progressive skin disease caused by Mycobacterium ulcerans, is prevalent in more than 30 countries, with especially high incidence in West Africa [1–4]. The source of M. ulcerans infection is still unknown; however, the bacteria has been detected in aquatic insects [5–8] and the disease mostly occurs in people who live near still water areas, suggesting that contaminated waters may act as a reservoir of M. ulcerans [5, 9].
In the early stages of the disease, papules, nodules, plaques, and edema are observed in the skin, followed by progressive ulceration eroding to subcutaneous layers and even bones [10, 11]. In extreme cases, patients may suffer contracture deformity and even amputation [2, 4]; therefore, early diagnosis and treatment is important for Buruli ulcer control. Common diagnostic methods include smear microscopy, histopathology, and culture; however, they have limited sensitivity or are time-consuming . Thus, it is necessary to develop simple and rapid tools that provide sufficient detection sensitivity to confirm the diagnosis of Buruli ulcer.
Several PCR methods for the detection of M. ulcerans have been reported; among them, the most widely used is based on targeting the IS2404 repeat sequence, which encodes a transposase and which is unique to M. ulcerans genome, where it occurs over 200 times [13–15]. Since other targets such as genes encoding 16S rRNA , hsp65  and the ketoreductase domain of mycolactone polyketide synthase [14, 18], or IS2606 [14, 18] have much lower copy numbers than IS2404, the sensitivity of their detection by PCR is lower than that of IS2404 [14, 15, 18]. Moreover, of these sequences, only IS2404 is specific for M. ulcerans [14, 15, 18]. IS2404 is also targeted by real-time PCR and loop-mediated isothermal amplification, which are more rapid and sensitive methods than gel-based conventional PCR and which have been recently applied for the detection of M. ulcerans [18–20]. Currently, real-time PCR is the gold standard method to confirm the presence of M. ulcerans [14, 15, 21].
Previous studies have used several primer sets for the amplification of IS2404 [14, 15, 22]; however, most of them were designed for gel-based conventional PCR  and may not be suitable for a more sensitive real-time PCR. Our in silico analysis indicates that the sequence of the IS2404 elements is highly variable and that the primers commonly used for IS2404 amplification are based on the polymorphic regions. Therefore, in this study, we designed and validated a new set of primers highly specific for stable sequences conserved among IS2404 copies as well as M. ulcerans strains with the aim to improve the sensitivity of M. ulcerans detection by real-time PCR.
Software for genetic analysis
Genetyx version 13 (Genetyx, Shibuya, Tokyo, Japan) was used for homology analysis and primer design. The alignment of IS2404 elements was performed by Genetyx version 13 using the algorithm of the MUSCLE program .
Bacterial strains and culture conditions
M. ulcerans strains and Mycobacterium species used in this study
Country of isolation
Mycobacteria were grown in Middlebrook 7H9 broth (BD Biosciences, Sparks, MD, USA) supplemented with 0.05 % (w/v) Tween 80 and 10 % (v/v) OADC Enrichment (BD Biosciences).
DNA extraction and purification
Bacteria were collected by centrifugation at 16,200×g for 2 min and resuspended in a solution containing 20 μl of 0.5 M NaOH, 4 μl of 10 % sodium dodecyl sulfate, and 180 μl of distilled water. The cell suspension was heated at 95 °C for 15 min, cooled to room temperature, and thoroughly mixed with 200 μl of phenol/chloroform (1:1). After centrifugation at 16,200×g for 5 min, the aqueous phase was transferred to a new tube and the extraction was repeated. Then, 20 μg of glycogen, 16 μl of 5 M NaCl, and 800 μl of 100 % ethanol were added to the pooled aqueous phases, and the mixture was centrifuged at 16,200×g for 15 min. The pellet was collected, mixed with 500 μl of 70 % ethanol, and the sample was centrifuged for 1 min. The final pellet was resuspended in 50 μl of distilled water.
Primers and probes for real-time PCR
Primer or probe
Statistical analysis was performed using the GraphPad Prism software version 6 (GraphPad Software, LA Jolla, CA, USA). The differences between samples were analyzed by the Student’s t test, and differences were considered statistically significant at a p value of 0.05.
IS2404 sequence variations
The genome of M. ulcerans strain Agy99 (gene accession number: CP000325) contains 249 IS2404-like elements . For the purpose of this study, 212 sequences with high homology to the transposase gene of IS2404 (MUL_0099) determined by BLASTN search were retrieved from the Agy99 genome and aligned with the transposase sequence used as a reference.
Sensitivity of the new primer/probe set for M. ulcerans detection
The use of the new primer/probe set enabled the detection of genomic DNA from most M. ulcerans strains using a smaller number of cycles than that required with the conventional set. PCR sensitivity was more significantly improved for three Japanese isolates (GTC16404, GTC16405, and GTC16406) than for Agy99 and TMC1615, while no changes were observed for ATCC19423 and ATCC33728. The results suggest that the new primer/probe set could improve the sensitivity of M. ulcerans PCR-based detection; however, the increase in sensitivity varied depending on the strain.
Specificity of the new primer/probe set
The IS2404-targeting PCR method, which enables rapid and sensitive detection of M. ulcerans, has been established as the gold standard for the diagnosis of Buruli ulcer [15, 18, 21] and is currently recommended by the WHO . IS2404 is a multi-copy insertion sequence encoding a 328-amino acid transposase , which is unique to M. ulcerans and is represented by 249 copies in its genome . Because of the high frequency of occurrence of IS2404 in the M. ulcerans genome, this element has been used as a target sequence in PCR-based detection of M. ulcerans infection. However, in this study, we revealed considerable sequence variability among the IS2404 elements of a single M. ulcerans strain (Agy99) (Fig. 1 and Additional file 1: Figure S1); nucleotide polymorphism was also observed in the regions targeted by commonly used primers, which could affect the accuracy of Buruli ulcer diagnosis. Therefore, to increase the detection sensitivity of M. ulcerans, we designed a new primer/probe set specific for the regions highly conserved among IS2404 copies. Compared with the conventionally used set, the new set provided an increased sensitivity and similar specificity of real-time PCR detection for most tested M. ulcerans strains (Figs. 2 and 3). Detection sensitivity with the new primer/probe set was particularly high for three Japanese isolates (GTC16404, GTC16405, and GTC16406); on the other hand, no changes were observed for the ATCC19423 and ATCC3372 strains. The results suggest that the new set could lead to better PCR-based detection of M. ulcerans than that with conventional primers, although the advantage may be strain dependent. In this study, we used only seven M. ulcerans isolates; more of them should be tested to comprehensively evaluate strain-specific differences in detection sensitivity using the new primer/probe set. In addition, it should be noted that clinical and environmental specimens could contain PCR inhibitors and contaminating DNA; therefore, the new set should be validated using a panel of clinical and environmental samples.
Since we observed sequence variability among IS2404 copies of the same M. ulcerans strain, we hypothesized that the difference may also exist among the strains. As described in results, PCR sensitivity was more significantly improved by new primers for three Japanese isolates. The result suggests that the Japanese strains could have more sequence diversity in the binding regions of conventional primers than other strains. In this study, we used M. ulcerans isolates from geographically distant areas. Interestingly, for the strains from Africa (Agy99), Southeast Asia (TMC1615), and Australia (ATCC19423), which belong to the classical lineage , the new primer/probe set provided only moderate or no improvement of detection sensitivity, while for most Japanese strains, a significant increase in sensitivity was observed. Weihong et al.  demonstrated higher frequency of large chromosomal rearrangements in a Japanese strain compared to the classical lineage strains . Since the IS2404-encoded transposase may be closely involved in genomic rearrangements, Japanese strains might harbor the IS2404 elements carrying different types of polymorphisms compared to the classical lineage strains. To clarify why the new primers improved the sensitivity of M. ulcerans PCR detection, further analysis, including whole genome sequencing of each strain may be required. On the other hand, there is possibility that particular M. ulcerans isolates may escape IS2404-targeting PCR detection. Therefore, to provide sensitive and robust detection of M. ulcerans, it might be useful to perform multiplex PCR, which would target, along with IS2404, several other M. ulcerans sequences such as IS2606 or ketoreductase domain in the genes encoding mycolactone polyketide synthase, as reported by Fyfe et al. .
Further sequence analysis of the IS2404 elements is necessary to develop more sensitive methods for M. ulcerans diagnosis.
The results of our study suggest that the new primer/probe set is more sensitive for PCR-based detection of M. ulcerans than the conventionally used set, suggesting that its application can improve the diagnosis of Buruli ulcer.
BHQ1, black hole quencher 1; FAM, fluorescein amidite; MGB, minor groove binder
We thank Ms. Sayuri Hishida and Ms. Eri Suzuki for the skillful technical assistance. We are also grateful to Dr. Small (University of Tennessee, USA) and Dr. Saito (Shimane University, Japan) for providing the mycobacterial strains. We would like to thank Editage (Tokyo, Japan) for the English language editing.
This study was supported by a grant from Kitasato University, Tokyo, Japan.
Availability of data and materials
The datasets supporting the conclusion of this study are included within the article and the supplemental data.
NS designed the study, analyzed the data, and wrote the manuscript. HN participated in the data analysis and manuscript preparation. MW assisted with the study design, data analysis, and manuscript preparation. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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