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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~ Acknowledgment
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 32  |  Issue : 3  |  Page : 236-239
 

Application of PCR fingerprinting using (GACA) 4 primer in the rapid discrimination of dermatophytes


1 Departments of Microbiology, Sri Ramachandra University, Porur, India
2 Department of Microbiology, Sri Muthukumaran Medical College, Hospital and Research Institute, Chikkralayapuram, Chennai, Tamil Nadu, India
3 Dermatology and Venereology, Sri Ramachandra University, Porur, India

Date of Submission25-Jul-2014
Date of Acceptance09-Oct-2013
Date of Web Publication10-Jul-2014

Correspondence Address:
A J Kindo
Departments of Microbiology, Sri Ramachandra University, Porur
India
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Source of Support: Funded by Sri Ramachandra University Chancellor Research Fellowship Grant,, Conflict of Interest: None


DOI: 10.4103/0255-0857.136548

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 ~ Abstract 

Background: Superficial fungal infections have a major impact on cosmetic health, affecting more than 20-25% of the global population, which is predominantly caused by dermatophytes. As per literature search, molecular strain typing of dermatophytes has not been investigated in India. Therefore, the present study was carried out to characterise the dermatophyte species and strains by molecular methods. Objective: To analyse the genotype variability by applying polymerase chain reaction (PCR) fingerprinting using a simple sequence repetitive oligonucleotide (GACA) 4 primer to identify the species and strain variations among the dermatophytes isolated from a tertiary care centre in Chennai. Materials and Methods: From January 2010 to December 2010, 81 dermatophytes were isolated and included for the present study. A simple sequence repetitive oligonucleotide (GACA) 4 was used as a single primer in the amplification process. Results: The (GACA) 4 -based PCR successfully amplified all the clinical isolates. Trichophyton rubrum and T. rubrum var. raubitschekii produced identical band profiles, where the latter could not be differentiated from the T. rubrum, which are being reported for the first time from south India. Epidermophyton floccosum produced species-specific band profiles. Intra-species variability was not observed among the T. rubrum and E. floccosum isolates. T. mentagrophytes produced three simple, distinct band patterns, which are surprisingly different from the earlier studies. Conclusion: The PCR-based genotype using the short primer is rapid and precise in direct identification of dermatophyte isolates by one-step PCR to the species level and strain discrimination of the T. mentagrophytes variants.


Keywords: Dermatophytes, (GACA) 4 primer, PCR fingerprinting


How to cite this article:
Elavarashi E, Kindo A J, Kalyani J, Sudha R. Application of PCR fingerprinting using (GACA) 4 primer in the rapid discrimination of dermatophytes. Indian J Med Microbiol 2014;32:236-9

How to cite this URL:
Elavarashi E, Kindo A J, Kalyani J, Sudha R. Application of PCR fingerprinting using (GACA) 4 primer in the rapid discrimination of dermatophytes. Indian J Med Microbiol [serial online] 2014 [cited 2020 Oct 28];32:236-9. Available from: https://www.ijmm.org/text.asp?2014/32/3/236/136548



 ~ Introduction Top


Dermatophytosis is a superficial mycoses caused by dermatophytes of the genera Trichophyton, Microsporum and Epidermophyton infecting the skin, hair and nails of humans and animals. They are the most frequently recognised organisms and over USD $500 million per year is invested for the drugs produced against dermatophytosis worldwide. [1] Few minor attributes influence the importance in identification of dermatophytes. Although many molecular-based analyses on the dermatophytes was developed earlier, yet there is always a fluctuation in the taxonomy, difficulty in naming of the pathogenic species and scientific queries on the strain differentiation are yet to be solved. Re-infection is often related to dermatophytosis, whether it occurs with the same dermatophyte species, a variant or a different species producing similar type of lesions. Anti-fungal drugs that are targeted against a dermatophyte species would obviously be effective against others as well. Thus, species identification may not rely on clinical treatment but still specific dermatophyte identification is necessary for the epidemiological concerns. Knowledge about the epidemiology plays a vital role in infection control and community health management. Epidemiological profile may differ due to certain factors like climatic conditions, environmental and socio-economic status and due to tourism. Therefore, to attain a classic picture of the taxonomy and the current epidemiological outbreak, the present molecular study on dermatophytes is significant.

Strain typing by phenotypic methods is practically difficult since the dermatophytes on sub-culture show morphological variations among the isolates. Molecular techniques have overcome certain limitations experienced by the routine conventional methods in the rapid identification of specific dermatophytes. Many molecular-based strain typing methods have been developed to discriminate the various species and strains such as restriction fragment length polymorphism (RFLP), arbitrary primed polymerase chain reaction (AP-PCR), random amplified polymorphic DNA (RAPD) analysis and sequence analysis of the internal transcribed spacer (ITS) region. [2],[3],[4],[5],[6],[7] Few techniques (RFLP, RAPD, AP-PCR) help to identify up to the species level, however, they exhibit limited sensitivity in the identification of strain variants. The information on identification of dermatophyte strain variations is few and moreover, there are no data from the Indian subcontinent where the molecular strain typing have ever been evaluated. Therefore, in the present study, an attempt was made to analyse the genotype variability by applying DNA fingerprinting using a simple sequence repetitive oligonucleotide (GACA) 4 primer to identify the species and strain variations among the dermatophytes isolated from a tertiary care centre in Chennai.


 ~ Materials and Methods Top


Clinical isolates

The study period was from January to December 2010. A total of 138 dermatological clinical specimens were randomly selected, who attended the Dermatology outpatient Department of a tertiary care centre in Chennai, of which 81 dermatophytes were isolated and utilised for the present study. Among the 81 clinical isolates, 53 were isolated from cases of tinea corporis, followed by 20 isolates from tinea cruris, 2 from tinea faciei, 1 from tinea unguium and 5 were isolated from a mixed infection of tinea corporis and tinea cruris. Standard ATCC strains of Trichophyton rubrum 28188 and T. mentagrophytes 9533 were used for the study. An ethical approval was obtained from the Institutional Review Board to conduct the study (IEC-NI/09/DEC/13/40). The sampling procedures were followed with ethical standards and the informed consent was obtained from the participants.

DNA extraction

The dermatophytes were sub-cultured on Sabouraud's dextrose agar (SDA) at 25°C for 2 weeks. The mycelium was collected and the genomic DNA was extracted following Omni PrepTM kit for fungus (G Biosciences, USA) according to manufacturer's instructions.

In brief, the mycelium was inoculated in 500 μl of genomic lysis buffer and was ground using mortar and pestle. Next 5 μl of proteinase K was added and incubated at 60°C for 2 hours. After adding 200 μl of chloroform, it was spun at 14000 rpm for 10 min. The supernatant was transferred to a new eppendorf and 50 μl of DNA stripping solution was added and incubated at 60°C for 10 min. A total of 100 μl of precipitation solution was added and was spun at 14000 rpm for 5 min. To the supernatant, ice-cold isopropyl alcohol was added to precipitate the DNA and washed twice with 700 μl of 70% ethanol, air dried, re-suspended with 80-100 μl of Tris-EDTA buffer and stored at − 20°C for future use.

PCR fingerprinting

The simple-sequence repeats (GACA) 4 was used as a single primer in the amplification process, which was utilised as described in an earlier study. [8] The primer and PCR reagents were purchased from Bangalore Genei Pvt. Ltd, Bangalore, India. The amplification was performed using Eppendorf MasterCycler Gradient Model - 5331 (Germany). The amplification was carried out for 40 cycles of denaturation at 93°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 1 min, followed by final extension at 72°C for 5 min. Each isolate was amplified in duplicates in the same PCR setup and they were repeated at different periods. Negative control (no template DNA) was also included in each PCR setup to confirm that the obtained bands are the amplified genomic DNA and not the primer artifacts. The amplicons were electrophoresed on 1% agarose gel, stained with ethidium bromide and visualised on Biorad XR (California, USA) gel documentation unit.


 ~ Results Top


In the present study, a short (GACA) 4 primer was utilised for the dermatophyte identification of 81 clinical isolates: T. rubrum (37), T. mentagrophytes (22), T. rubrum var. raubitschekii (10), T. interdigitale (3) and Epidermophyton floccosum (9). The band pattern obtained were simple, producing four to six bands ranging from 300 to 1900 bp size. The PCR product band patterns using the simple primer are summarised in [Table 1].
Table 1: The PCR product band patterns using (GACA)4 primer


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All T. rubrum isolates produced similar band pattern, which consisted of three bright bands (approximately 600, 1000, 1100 bp) and one faint band at 1900 bp, which are depicted in [Figure 1]. T. rubrum var. raubitschekii isolates produced identical band profiles of T. rubrum. All E. floccosum isolates produced identical band profiles, which consisted of five bright bands size ranging approximately between 300 and 800 bp, and one faint band at 1100 bp [Figure 2]. For the T. mentagrophytes isolates (22 T. mentagrophytes and 3 T. interdigitale isolates), three profiles were observed, which are depicted in [Figure 3]. The first profile (T. mentagrophytes isolates - Type I) consists of three bright bands approximately at 300, 600 and 1500 bp [Figure 3]: Lane 2 and 3]. The second profile (T. mentagrophytes isolates - Type II) consists of four bright bands approximately at 300, 600, 800 and 1500 bp [Figure 3]: Lane 4 and 5]. The third profile (T. interdigitale isolates - Type III) consists of four bright bands at 300, 600, 650 and 1500 bp [Figure 3]: Lane 6 and 7]. Eighteen were Type I, four were Type II and three were Type III of T. mentagrophytes complex.
Figure 1: PCR fingerprints of T. rubrum complex. Lane 1: Negative control; Lane 2: T. rubrum ATCC 28188; Lane 3-5: T. rubrum clinical isolates; Lane 6 and 7: T. rubrum var. raubitschekii clinical isolates; Lane 8: GeNei Low range DNA ruler

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Figure 2: PCR fingerprints of E. floccosum. Lane 1: Negative control; Lane 2-7: E. floccosum clinical isolates; Lane 8: GeNei Low range DNA ruler

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Figure 3: PCR fingerprints of T. mentagrophytes complex. Lane 1: Negative control; Lane 2 and 3: T. mentagrophytes clinical isolates - Type 1 pattern; Lane 4 and 5: T. mentagrophytes clinical isolates - Type 2 pattern; Lane 6 and 7: T. interdigitale clinical isolates - Type 3 pattern; Lane 8: T. mentagrophytes ATCC 9533; Lane 9: GeNei Low range DNA ruler

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 ~ Discussion Top


Molecular typing is rapid and sensitive as compared with the phenotypic characterisation of dermatophytes to the species and strain levels. Moreover, these techniques are dependent on the genetic makeup, which would be more accurate than the conventional confirmations. In our previous work, we compared the culture method and two PCR-based RFLP targeting the ITS and 18S rDNA region using three restriction enzymes and identified the dermatophytes up to the species level. No intra-specific variations were observed. [2] Therefore, for the identification of strain variants, in the present study, we used a simple repetitive oligonucleotide (GACA) 4 primer, which is found to be useful in the rapid identification of dermatophyte isolates and T. mentagrophytes strain variants.

In our study, we successfully amplified all the clinical isolates and the band pattern obtained gave a classic picture between dermatophyte species. A well-marked similarity was recognised between the band profiles of T. rubrum and T. rubrum var. raubitschekii isolates in the present study. Therefore, the latter could not be differentiated from T. rubrum strains, which are being reported for the first time from south India. Since these two species are closely related, it must be considered under T. rubrum complex. [9] Species-specific band pattern was observed among the E. floccosum isolates. However, no intra-specific variation was recognised among the T. rubrum and E. floccosum isolates. Therefore, in our study, the band profiles obtained for the T. rubrum and E. floccosum isolates were in accordance with the earlier study. [8] The T. mentagrophytes isolates produced three distinct band profiles, of which the second and third band profiles differed from the first profile by a single band size approximately at 800 and 650 bp, respectively. The band patterns of T. mentagrophytes isolates consisted of three or four bands, which are surprisingly different from the earlier studies, producing complex band profiles of the T. mentagrophytes variants. [8],[10]

The PCR reaction mixture (dNTP mix, primer, taq buffer, taq enzyme and genomic DNA) were prepared and stored at -20°C. On the 5 th day the amplification was carried out to test the stability of the primer. We observed that the primers were not degraded even after 5 days of cold storage. The band patterns of the clinical isolates were also identical to the band profile obtained when the amplification was carried out on the same day.

In general, dermatophytes easily lose their specific morphological features in culture. Recently, few methods like real-time PCR and MALDI-TOF mass spectrometry have been optimised for the rapid identification of the dermatophyte species. [11],[12] The advantage of these methods are comparatively more than the phenotype identifications, however, the main disadvantage is the higher cost and may not be affordable for the routine laboratory settings.

The identification of the genotype variations using the short (GACA) 4 primer gives a classic discrimination between the T. rubrum and T. mentagrophytes by a single-step PCR. As reported in earlier studies, PCR-based fingerprinting using (GACA) 4 primer is simple and rapid in identification of dermatophyte isolates and T. mentagrophytes variants. [8],[10],[13]


 ~ Conclusion Top


Our analysis on molecular typing using the (GACA) 4 primer was used for the first time from south India to identify the genotype variations among the dermatophytes isolated from a tertiary care centre in Chennai. PCR fingerprinting is rapid as it takes only about 7 hours to identify the dermatophyte species from cultures, whereas the standard conventional laboratory identification takes about few weeks. The (GACA) 4 -based PCR is simple, efficient and reliable method in identification of dermatophyte species and in strain typing of T. mentagrophytes variants. Intra-species variation was not detected among T. rubrum and E. floccosum. Hence, the application of PCR fingerprinting using the simple repetitive primer is a cost effective method and therefore, can be applied for the routine laboratory practices.


 ~ Acknowledgment Top


We thank Sri Ramachandra University for the award of SRU - Chancellor Research Fellowship Grant to conduct the study at Sri Ramachandra University, Chennai, India.

 
 ~ References Top

1.Gräser Y, Scott J, Summerbell R. The new species concept in dermatophytes-a polyphasic approach. Mycopathologia 2008;166:239-56.  Back to cited text no. 1
    
2.Elavarashi E, Kindo AJ, Kalyani J. Optimization of PCR-RFLP Directly from the Skin and Nails in Cases of Dermatophytosis, Targeting the ITS and the 18S Ribosomal DNA Regions. J Clin Diagn Res 2013;7:646-51.  Back to cited text no. 2
    
3.Rezaei-Matehkolaei A, Makimura K, Shidfar M, Zaini F, Eshraghian M, Jalalizand N, et al. Use of Single-enzyme PCR-restriction Digestion Barcode Targeting the Internal Transcribed Spacers (ITS rDNA) to Identify Dermatophyte Species. Iran J Public Health 2012;41:82-94.  Back to cited text no. 3
    
4.Liu D, Coloe S, Baird R, Pedersen J. Molecular determination of dermatophyte fungi using the arbitrarily primed polymerase chain reaction. Br J Dermatol 1997;137:351-5.  Back to cited text no. 4
    
5.Baeza LC, Matsumoto MT, Almeida AM, Mendes-Giannini MJ. Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer. J Med Microbiol 2006;55:429-36.  Back to cited text no. 5
    
6.Gräser Y, Kuijpers AF, Presber W, De Hoog GS. Molecular taxonomy of Trichophyton mentagrophytes and T. tonsurans. Med Mycol 1999;37:315-30.  Back to cited text no. 6
    
7.Li HC, Bouchara JP, Hsu MM, Barton R, Su S, Chang TC. Identification of dermatophytes by sequence analysis of the rRNA gene internal transcribed spacer regions. J Med Microbiol 2008;57:592-600.  Back to cited text no. 7
    
8.Faggi E, Pini G, Campisi E, Bertellini C, Difonzo E, Mancianti F. Application of PCR to distinguish common species of dermatophytes. J Clin Microbiol 2001;39:3382-5.  Back to cited text no. 8
    
9.Gräser Y, Kuijpers AF, Presber W, de Hoog GS. Molecular taxonomy of the Trichophyton rubrum complex. J Clin Microbiol 2000;38:3329-36.  Back to cited text no. 9
    
10.Shehata AS, Mukherjee PK, Aboulatta HN, el-Akhras AI, Abbadi SH, Ghannoum MA. Single-step PCR using (GACA) 4 primer: Utility for rapid identification of dermatophyte species and strains. J Clin Microbiol 2008;46:2641-5.  Back to cited text no. 10
    
11.Yüksel T, Ilkit M. Identification of rare macroconidia-producing dermatophytic fungi by real-time PCR. Med Mycol 2012;50:346-52.  Back to cited text no. 11
    
12.Nenoff P, Erhard M, Simon JC, Muylowa GK, Herrmann J, Rataj W, et al. MALDI-TOF mass spectrometry: A rapid method for the identification of dermatophyte species. Med Mycol 2013;51:17-24.  Back to cited text no. 12
    
13.Khosravi AR, Shokri H. Rostami A, Tamai IA, Erfanmanesh A, Memarian I. Severe dermatophytosis due to Trichophyton mentagrophytes var. interdigitale in flocks of green iguanas (Iguana iguana). J Small Anim Pract 2012;53:286-91.  Back to cited text no. 13
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
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