Development of a Rapid and Low-Cost Method for the Extraction of Dermatophyte DNA.

Background: Polymerase chain reaction (PCR) is the most optimized method for the rapid detection and analysis of any environmental or clinically significant organism. While PCR amplification directly from samples has been shown effective for several bacteria and viruses, for filamentous fungus and yeast, extraction of genomic DNA is a must. The extraction of DNA from fungal cultures is often reported using user-friendly commercially available kits, which are designed to decrease the time, extensive manual work in extraction procedures but are often expensive. Dermatophytes pose an added drawback to efficient DNA extraction due to their poor recovery on culture media and slow growth rate. Aims and
Objectives: In the present study, we developed and validated a method for effective genomic DNA extraction from dermatophytes. Materials and
Methods: DNA yield from standard dermatophytes extracted from spore suspensions and mycelia mat by commercially available kits was compared. A modified method using lyticase buffer and phenol-chloroform extraction was developed. The yield obtained was compared with the existing methods (kit-based method and cetyl trimethyl ammonium bromide method). The yield and quality of the total genomic DNA were estimated spectrophotometrically and by successful PCR amplification of the ITS region. The results were validated using 21 clinical isolates from recalcitrant dermatophytosis.
Results: Minimal fungal DNA was obtained from the spores compared to that obtained from mycelial mat. Commercially available kits yielded lower amounts of DNA compared to the CATB method. The modified method developed in this study yielded better quality and quantity of DNA.
Conclusion: Of the three extraction methods evaluated, the developed method gave significantly higher total genomic DNA yield and better purity than the reference methods. In addition, the turnaround time for DNA extraction was reduced to half based on modifications in culture conditions. Copyright:

Introduction

Fungi are a large group of eukaryotic organisms, most of which are saprobes in soil and take part in the decomposition of organic matter. Extensive study interests in this group have risen due to their clinical importance, environmental function, antibiotic production, and role in food production.[1] Fungal infections are due to invasion of host tissue by fungal elements or detrimental effects of their metabolic products. Superficial fungal infections in humans (dermatophytosis) have risen in numbers over the recent years. The prevalence of dermatophytosis in India was reported as 36.6%–78.4% in 2018.[23] Identification of the causal agent of these infections has traditionally been based on morphological characterization using specialized media for isolation, which requires close to 6 weeks or more for the appearance of distinct colonies.[45] The use of molecular techniques has become extensively crucial in many areas of biology, including detection, identification, epidemiological studies, and genetic mapping of several organisms (including fungus) as they are more rapid and sensitive.[6]

Molecular detection of any organism requires that the genomic DNA template be extracted preferably by fast and simple methods. DNA extraction methods for bacteria are well studied, which, when used for fungal DNA extraction, show reduced recovery due to the fact that the fungal cell wall is composed of chitin and polysaccharides such as glucan and mannan, which renders rigidity and complexity to the cell structure.[78] Most often, DNA extraction for fungus has been reported using expensive commercially available kits. Kits are designed to decrease the complex extraction procedures, ideal for researchers who are familiar with the methods, and with a small number of samples. However, the expenses incurred for purchase are very high, and regular testing laboratories may not be able to afford the same. There are many conventional fungal DNA extraction procedures that are time-consuming and often involve mechanical destruction, such as grinding,[910] freeze-drying,[11] magnetic beads,[12] microwave,[13] and enzymatic digestion[1415] for primary disruption of the cell. The present study aimed to develop and compare fungal DNA extraction methods to obtain good quality and sufficient quantity DNA necessary for amplification by conventional thermocycling.

Materials and Methods

Fungal cultures and culture conditions

Standard cultures of Trichophyton mentagrophytes ATCC9533, Microsporum canis ATCC36299, Trichophyton tonsurans ATCC28942, Trichophyton rubrum ATCC 28188 (procured through HiMedia), and known clinical isolates of Epidermophyton floccosum and Microsporum gypseum were used as the internal controls for all tests conducted.

Saprophytes, including isolates of Rhizopus spp., Penicillium spp., and Aspergillus niger, were isolated from the environment on Sabouraud's dextrose agar (SDA). Pure cultures of the same were subcultured in Sabouraud's dextrose broth (SDB) for 5 days at 27°C.

Clinical cultures from patients with dermatophytosis were cultured on SDA (used for phenotypic identification) and simultaneously grown in broth supplemented with 50 mg/ml cycloheximide, and 40 mg/ml chloramphenicol. SDA was incubated at 27°C for up to 4 weeks, while SDB was incubated in a shaker at 120 rpm at 27°C for up to 4 weeks.

For DNA extraction from cultures grown on SDA, the plate was flooded with fungal saline and swirled to obtain a conidial suspension. The suspension was then transferred to a tube and centrifuged at 6500 g for 10 min to collect the conidial pellet. The pellet thus obtained was weighed out and used for DNA extraction.

Alternatively, for cultures that grew in SDB, the culture was centrifuged at 6500 g for 10 min to collect the fungal mass. Appropriate mass was weighed out and used for DNA extraction.

DNA extraction methods

For the fungal cultures grown on SDA, the surface was flooded with fungal saline and scrapped. This suspension was centrifuged at 6000 g for 10 min and the pellet was used as fungal mass. In the case of cultures grown in SDB, 1 ml of the culture was centrifuged at 6000 g for 10 min and the pellet was used as fungal mass.

DNA extraction by cetyl trimethyl ammonium bromide method

Fungal mass was ground in a mortar with liquid nitrogen.[16] The cetyl trimethyl ammonium bromide (CTAB) extraction buffer (2% CTAB, 20-mM EDTA, 1-M NaCl, 100-mM Tris HCl), was added, and three rounds of freeze-thawing cycles were done. To the resultant solution proteinase K (20 mg/ml) was added and incubated at 60°C for 90 min. Extraction with phenol-chloroform isoamyl alcohol (25:24:1) and precipitation with isopropanol were performed. The alcohol precipitated DNA was dissolved in 30 μl of nuclease-free water.

Spin column method

Fungal DNA extraction using a Qiagen DNA extraction kit was performed as per the manufacturer's instructions.[17] The fungal suspension was incubated at 37°C for 60 min with lyticase extraction buffer (1-M Tris HCL, 0.5-M EDTA, β-mercaptoethanol, 10-U/μl lyticase). This was followed by proteinase K treatment at 56°C till complete lysis with ATL buffer (~60 min). The suspension was purified in an affinity spin column. The DNA was eluted in 30 μl of elution buffer.

DNA extraction by the modified method

Fungal mycelia were disrupted by incubating at 37°C for 30 min with lyticase extraction buffer (1-M Tris HCL, 0.5-M EDTA, β-mercaptoethanol, 10-U/μl lyticase), followed by incubating at 60°C for 60 min with lysis buffer (10-mM Tris, 10-mM KCl, 10-mM MgCl2, 0.5 M NaCl, 2-mM EDTA, 0.5% SDS) and proteinase K (20 mg/ml). After the incubation, extraction with phenol-chloroform isoamyl alcohol (25:24:1) and precipitation with isopropanol were performed. The DNA was eluted in 30 μl nuclease-free water.

The quality and quantity of DNA extracted by the three methods were measured using a nanodrop spectrophotometer. The average time required for extracting DNA by each of the methods was recorded.

PCR for ITS region

To further determine the quality of DNA extracted, the DNA was added as a template for amplification of fungal ITS region using primers targeting universal barcode internal transcribed region for fungus.[15] The reaction mixtures (30 μl) were set up as follows: 3 μl of 10X buffer (100-mM Tris HCl, 1.5-mM of MgCl2), 2.5-nM concentrations each of deoxyribonucleotide phosphates (dATP, dTTP, dGTP, dCTP), 10 picomoles of each primer ITS 1 (F-5'-TCCGTAGGTGAACCTTGCGG-3') and ITS 4 (R-5'-TCCTCCGCTTATTGATATGC-3') and 1-U of Taq DNA polymerase (Himedia Laboratories Pvt. Ltd., India), ~100 ng of template DNA and volume made up to 30 μl with nuclease-free water. The reactions were carried out in a thermocycler (Eppendorf NexusGX2) with the following conditions: 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 47°C for 30 s, 72°C for 30 s, followed by final extension at 72°C for 10 min. PCR products were resolved by electrophoresis on 2% agarose gel, stained with SYBR safe, and bands were visualized using a gel documentation system.

Results

Fungal identification: A total of 21 clinical isolates were considered for the study. These isolates were recovered from clinical samples inoculated in both SDA and SDB. Phenotypically, 17 isolates were identified as Trichophyton mentagrophytes and four were identified as Microsporum gypseum.

Visible growth of dermatophytes sufficient for DNA extraction was observed after 14 days on SDA, while sufficient growth in broth was observed within 5 days.

Minimal fungal mass from SDA when compared to fungal mass obtained from SDB at day 5 of incubation was observed. DNA thus extracted from the low mass also yielded low DNA concentration [Table 1]. Hence, for further studies, DNA recovery from broth cultures by the three methods was compared. The fungal mass obtained from SDB was divided into three portions, each of approximately 100 mg (accurate weight recorded), and DNA was extracted simultaneously by the three different methods. The comparison of yields obtained from 30 samples using CTAB, spin column method, and lyticase is illustrated [Figure 1 and Table 2]. The yield obtained by the modified method is significantly greater than the other two methods in terms of quantity (ANOVA: P = 0.01 when the modified method was compared with the CTAB method and P = 0.000074 when the modified method was compared to the kit method).

Table 1

DNA concentration in μg/μg mass of fungal spore suspension

Samples	Spin column	CTAB
1	0.2655	0.507
2	0.0915	0.906
3	0.159	0
4	0.0105	0
5	0.195	0
6	0.213	0

Figure 1

Comparative yield of DNA extracted using different methods. The concentration of DNA for 87% (27/31) isolates extracted by the modified method (Square data points) cluster above 100-ng DNA/ μg of fungal mass. DNA yield by the spin column method (Circular data points) was above 100 ng/ μg for 33% (7/31) and 58% (18/31) samples yielded DNA more than 100 ng/ μg fungal mass extracted by the CTAB method

Table 2

DNA concentration in μg/μg of fungal mass from SDB

Samples	SPIN COLUMN		CTAB		Modified
	Conc.	260/280	Conc.	260/280	Conc.	260/280
Aspergillus niger	118.8	1.8	136.09	2.0	149.71	1.9
Rhizopus spp.	17.72	2.0	32.089	1.8	36.213	1.2
Penicillium spp.	45.15	1.7	480.00	2.1	618.05	1.9
T. mentagrophytes ATCC9533	17.145	2.1	82.092	2.1	701.1	1.8
T. rubrum ATCC28188	8.715	3.0	26.679	2.4	340.63	1.9
M. canis ATCC36299	14.52	2.2	286.24	2.1	857.47	1.5
M. gypseum	112.90	2.2	129.01	2.2	256.75	1.6
E. floccosum	30	2.7	1025.8	1.9	489.79	1.5
T. tonsurans ATCC28942	46.2	2.5	101.9	2.2	332.6	1.7
Sample 1 T. mentagrophytes	85.878	2.1	236.54	2.0	667.47	1.7
Sample 2 T. mentagrophytes	29.418	2.2	174.05	2.0	224.14	1.6
Sample 3 M. gypseum	4.368	2.1	19.62	2.1	34.125	1.9
Sample 4 T. mentagrophytes	116.1	1.8	180.88	2.2	382.03	1.8
Sample 5 M. gypseum	24.756	1.8	116.75	1.9	170.5	1.7
Sample 6 M. gypseum	50.166	2.0	440.89	2.0	681.47	1.8
Sample 7 T. mentagrophytes	89.232	2.1	99.768	2.1	187.37	1.8
Sample 8 T. mentagrophytes	90.081	2.1	126.31	2.2	125.49	1.8
Sample 9 T. mentagrophytes	4.242	2.1	88.89	2.1	297.19	1.9
Sample 10 T. mentagrophytes	230.69	2.1	321.96	2.0	396.97	1.8
Sample 11 T. mentagrophytes	67.641	1.9	98.7	2.0	1074.3	1.7
Sample 12 T. mentagrophytes	32.076	1.8	148.41	2.1	152.29	1.7
Sample 13 T. mentagrophytes	50.145	2.0	260.29	2.0	476.34	2.0
Sample 14 T. mentagrophytes	120.99	2.6	171.47	2.1	211.05	1.8
Sample 15 T. mentagrophytes	64.734	2.2	100.14	2.2	194.12	1.5
Sample 16 M. gypseum	55.485	2.4	120.40	2.1	228.77	1.7
Sample 17 T. mentagrophytes	31.287	2.1	47.379	2.9	35.259	1.7
Sample 18 T. mentagrophytes	30.441	2.2	61.485	1.8	346.94	1.8
Sample 19 T. mentagrophytes	57.339	2.1	135.51	2.8	92.595	1.6
Sample 20 T. mentagrophytes	56.427	1.8	96	2.6	175.81	1.8
Sample no 21 T. mentagrophytes	43.092	1.8	50.349	2.2	175.81	1.9

The wholeness and the quality of fungal genomic DNA extracted were validated using PCR. With the DNA extracted by the modified method, all the samples subjected for amplification using the primers targeting internal transcribed spacer region, single bands of ~700 base pairs were obtained.

Discussion

The identification of the causal agent of dermatophytosis is of epidemiological importance; this not only reflects the etiological agent but also has implications on treatment strategies.[3] Though the gold standard for identification relies on culture retrieval from a clinical specimen, the poor diagnostic sensitivities (<40% recovery) and long turnaround times (up to 14 weeks) have paved the way for non-cultivation methods to overcome these drawbacks. A major setback to molecular detection of dermatophytes is the fungal nucleic acid extraction from skin scrapings. In general, DNA from filamentous fungus is more difficult to extract than that of other microorganisms due to the complex and rigid structure of the fungal cell wall. Breaking this cell wall using the conventionally available extraction methods employed for other microorganisms is not applicable. Poor efficiency of DNA extraction procedures tends to give a low concentration of DNA, which in turn is the reason for acquiring false-negative results in any molecular analysis. Therefore, along with using a specific primer for the detection of the fungus in low concentrations, it is equally important to use the most effective method of DNA extraction.[18] A variety of methods have been described for genomic DNA extraction of fungus, though many of these are not applicable for a laboratory where many samples are to be analyzed simultaneously.[19] Loss of fungal DNA during the extraction process is a major limitation to molecular diagnostic tools for pathogenic fungi.[20] The clinical specimen recovered in certain conditions of dermatophytosis is also very low to yield sufficient DNA to be directly amplified by PCR. Hence, some form of culturing is often required to supplement non-cultivation methods to increase the initial template levels. Alternatively, nested PCR has been used to increase sensitivity; however, this too increases the cost of detection and requires a large volume of template DNA (15 μl) to be added in the first cycle of amplification.[20] In our study, we reduced the time required for fungal mass recovery by culturing the specimen in SDB and incubating at 27°C, 120 rpm for 5 days. Further incubation up to 7 days increases the fungal mass and hence increased DNA yield, but an incubation limited to 5 days yielded sufficient mycelial mass for good DNA yield. In our study, it was observed that fungal mass from SDA was minimal compared to that recovered from SDB in the same incubation period. In the case of fungal mass removal from solid media, only surface elements like conidia and aerial hyphae come into the fungal saline while the mycelial mat within the agar is not recovered. However, in the case of the SDB that was incubated with shaking, the entire fungal mass settled at the bottom of the tube and was recovered for DNA extraction. Gnat et al.[21] reported better DNA yield on solid medium in 7 days compared to that in the broth while our study suggests otherwise. A probable explanation for higher yield in SDB in our study is that while previous studies report the nucleic acid extraction from conidia (only from the surface mat), we used vegetative fungal hyphae, which develop in the broth. The cell wall of spores is known to withstand harsh conditions that are used in cell denaturation while the vegetative hyphae may be easier to penetrate and damage.[22] Pretreatment of sample material with lytic enzymes is required for sensitive DNA extraction irrespective of manual or automated systems of fungal nucleic acid extraction.[14] In the modified protocol reported in this study, we used two-stage enzymatic lysis, first cell wall lysis with lyticase and then protein lysis with proteinase which had resulted in relatively uniform DNA yield from dermatophytes. Cell lysis followed by phenol-chloroform extraction is the most traditional method of DNA extraction which digests the cell materials and removes the contaminants by using organic solvents. DNA extracted by this method can be preserved for several years at −20°C.[23] Methods that report phenol-chloroform extraction used freezing with liquid nitrogen and grinding for cell lysis, which limits the use of these methods to laboratories equipped to store liquid nitrogen. Innovative technologies, like bashing beads, provide for rapid DNA extraction; however, this too requires the procurement of the ultra-high density lysis matrix, fracture-resistant lysis tubes, and Bead beater, which incur costs.[24] Automated DNA extraction systems have less turnover time, are more sensitive, and provide DNA of better purity. However, this requires the installation of high-end equipment, which is not feasible for small-scale laboratories.[2526] In our study, the spin column kit method failed to extract amplifiable DNA from some fungal isolates, which is in consensus with previous reports.[212627] The best amplifiable DNA was observed in the modified method. We could successfully amplify the internal transcribed gene, which has been considered as the universal barcode for fungus as evident upon gel electrophoresis, which confirmed that the extracted DNA can be used for many molecular techniques. This novel method for fungal genomic DNA extraction yields better results and can be considered to be more effective in terms of time, cost, and yield [Table 3].

Table 3

Qualities of the three methods compared for fungal DNA extraction

Extraction method	Cost per reaction in Indian rupees (reagents and labware only)	Time	Average yield (ng/μg fungal mass)	Reagents	Equipment
Modified method (this study)	~50/-	~2 h	342.6	Lyticase buffer, proteinase K, PCIA	Dry bath, centrifuge
CTAB	~50/-	~3 h	185.5	Liquid nitrogen, CTAB buffer, proteinase K, PCIA	Dry bath, centrifuge, mortar grinder
Kit	~200/-	~3 h	57.5	Commercial kit and specific reagents	Dry bath, centrifuge

Financial support and sponsorship

The authors are grateful to Nitte (Deemed to be University), Mangalore for funding this work via grant numbers NUFR1/2018/10/06 and NUFR2/2018/10/14.

Conflicts of interest

There are no conflicts of interest.