Abstract

Over 200 species of fungi are responsible for a variety of infections that can occur in various parts of the human body. There are several phenotypic methods for identifying these fungal elements; however, these approaches have limitations. Molecular methods are now routinely used in well-equipped mycology laboratories. However, the first step in isolating genetic material can often be costly, can suffer from external DNA contamination and some components have toxicity for personnel. The general objective of this work was to identify the best local method for isolating genetic material from fungi based on cost, yield and time-consuming criteria. A total of ten (10) different nucleic acid isolation methods were tested. Those tests using thermal or mechanical shock for cell lysis delivered better quality than those using chemical lysis. Thus, based on our criteria, the best methods for nucleic acid isolation and purification of fungal elements were cetyltrimethylammonium bromide (CTAB) combined with sterilized sea sand (less expensive) and the chelator Chelex® coupled with glass beads (faster).

1. Introduction

Mycoses are fungal infections caused by microscopic fungi with a filamentous or yeasty appearance. They have been a major public health issue since the 1980s ¹. Routine laboratory identification at the Pasteur Institute of Côte d'Ivoire is done phenotypically. However, these phenotypic methods have limitations. Indeed, they allow the identification of a certain number of yeasts by chromogenic areas and do not identify in an absolute manner through semi-automated or automated methods such as VITEK, MALDI-TOF ² etc. Considering these limitations, molecular biology approaches present alternative or complementary methods for detecting, identifying and typing fungal pathogens. Pneumocystis PCR is on the WHO EDL (Essential Diagnostics List), as a reason for doing the work and many other molecular tests (Candida in blood, Candida auris on surveillance swabs (OLM Diagnostics®), Aspergillus PCR (including species and/or resistance (Pathonostics®, OLM), Trichophyton and Mucorales are available.

However, in order to use molecular methods, several important steps are necessary. The first crucial step is the purification of genetic material. This phase consists of three (3) major steps: cell lysis, purification of genetic material, and elution into an appropriate conservation buffer ³. The lysis step in the case of fungal elements is more complex due to the richness of the cell membrane in polysaccharide ⁴. Various commercial kits are available to circumvent this problem but, in some cases, the high cost makes them inaccessible for routine use. In order to avoid commercial kits, various methods have been tested by several research teams. Chemical cell lysis is commonly used, although these products can be toxic to the human body ^{5, 6, 7, 8}. Beads ⁸ or enzymatic digestion ^{9, 10} are also viable methods for cell lysis. Membrane or silica matrix applications are also used to recover nucleic acids ^{11, 12}.

The general objective of our work was to test various local methods for isolating genetic material from fungal elements in order to identify the best approach according to cost, yield and duration criteria. In this work, tested three methods for cell lysis of fungal material (chemical, thermal or mechanical means) and several approaches for isolation and purification of DNA (Chelex® 100, CTAB and modified Qiagen® cells and tissus DNeasy protocol).

2. Material and Methods

Single cultures of Candida albicans and Aspergillus fumigatus were obtained from clinical samples of patients at the Pasteur Institute of Côte d'Ivoire (Table 1). After freeze preserving in Brain heart broth or Bouillon Coeur cervelle (BCC) + glycerol, they were grown on Sabouraud media solid agar for 2 days for Candida albicans and 5 days for Aspergillus fumigatus. After culture, mycelium were transferred to a locking microcentrifuge tube with 300µl of sterile Phosphate Bovine Saline (PBS) 1X to obtain 0.5 McF opacity suspension.

Three different lysis protocols are described here: (i) thermal lysis, (ii) physical lysis, (iii) chemical lysis. For thermal lysis, suspensions were mixed and placed in liquid nitrogen for 30 minutes while thawing every 5 minutes in a liquid water bath at 100°C. For mechanical lysis, suspensions were transferred to a locking microcentrifuge tube containing glass beads or sterilized sea sand. Lysis was achieved by vortexing for 30 min at the highest intensity setting utilizing a Bead-Bug for 1 min 30 sec. For chemical Lysis, 200 µL of suspension were transferred in 200 µL of CTAB buffer (100 mM Tris pH 9; 1.4 M NaCl; 20 mM EDTA; 2% (w/v) hydroxycetyl trimethylammonium bromide (CTAB); 0.2 % (v/v) β-mercaptoethanol) or ATL lysis buffer Qiagen, vortexing then placed in water bath at 65 °C for 30 min.

The lysate obtained from each lysis method was used for nucleic acid purification.

To remove residual cellular debris, 250 µL of Phenol/Chloroform/Isoamyl Alcohol solution was added to the lysate and the microtube was centrifuged for 5 minutes at 15000 rpm and the supernatant transferred to a new microtube. Two volumes of absolute ethanol and 1/10 of Sodium acetate 3M were added to the supernatant, and the microtube was centrifuged for 20 min (15000 rpm). DNA was precipitated with 1 mL of 70% ethanol (centrifugation for 20 mn, 15000 rpm). The ethanol was removed and the pellet was dried at room temperature.

To use Chelex protocol, 250 µL of Chelex 5% previously activated by heating to 100°C in a water bath for 5 min, and 20 µl of proteinase K was added to the lysate. The microtube was incubated at 65°C for 30 min. To remove proteins debris, the microtube was spun at 12000 rpm for 2 min. The supernatant was transferred in new microtube and spun at 12000 rpm for 2 min. The supernatant was transferred again in new microtube for storage at -20°C.

This procedure was employed with sterilized sea sand, glass beads and thermal lysates. The purification started by add 800 µL of CTAB buffer previously heated at 65°C for 5 min in the water bath following by vortexing for 20-30 seconds. The microtube was incubate at 65°C for 30 minutes and continuous vortexing every 10 minutes; following by addition of 800 µL of Chloroform/Iso Amyl Alcohol (24:1), vortexing for 30-60 seconds and centrifuged at 13000 rpm for 10 minutes. The supernatant was transferred to a new microtube and mixed with 500 µl isopropanol then incubated at -20°C for 2 hours or overnight. After incubation, the microtube was centrifuged at 13000 rpm for 10 minutes and the pellet was transferred again in new microtube. DNA was washed by adding 200 µl of 70% ethanol to the supernatant and centrifuged at 13000 rpm for 5 minutes. The purification was achieved by removing ethanol and the pellet was dried at room temperature.

The lysates were centrifuged at 1880 RPM for 5 min and the supernatant was transferred to a new 2 ml Eppendorf tube. The purification continued following the Qiagen protocol.

Proteins were removed by transferring the lysate supernatant to CTAB in a new 1.5 ml Eppendorf tube after extraction with 200 µl of chloroform mixed by inversion and centrifuged at 12000 RPM for 5 min. 200 µl of isopropanol was added and mixed via inversion, followed by centrifugation at 12,000 RPM for 15 min to separate and wash the DNA from the CTAB Buffer. The DNA was precipitated by 200 µl of 70% ethanol by centrifugation at 12000 RPM for 5 min. The ethanol was removed and the extract was left at room temperature to dry the pellet.

To the lysate with ATL Qiagen, 200 µl of Chelex 5% previously activated by heating to 100 °C in a water bath for 5 min was added. Next, the mixture was heated to 100 °C to perform cell lysis. 20 µL of proteinase K was added and incubated at 65 °C for 30 min to activate the enzyme and lyse the proteins. Residual proteins were removed by centrifugation at 12000 RPM for 2 min. The remaining elements were removed by centrifuging the supernatant for 2 min at 12000 RPM.

All purified products were eluated in Qiagen AE buffer with the exception of the DNA isolated using Chelex (products obtained after purification were stored directly at -20 °C as nucleic acid extract).

Extraction methods are summarized in Table a.

The origins of the different isolates and strains used in this study are listed in the table below

The PCR solutions were prepared with purified template; and each target was amplified according the appropriate programs.

Strip size was evaluated by a visual reading of 8 µl on a 1% agarose gel for 45 minutes at 100V.

The table below shows the different volumes to be taken of each reagent and the necessary concentrations of enzyme and primer in the reaction medium.

The different times required for each amplification step for each primer pair are listed in the table above.

3. Results

A total of ten (10) fungal DNA extraction methods were tested: four (4) which included mechanical tests, four (4) which included thermal tests, and two (2) which based on chemical products for lysis of fungal cells (Table 1). The PCR results were compared between the types of methods (lyses) of the different protocols. Figure 1 shows the results obtained with the identification primers Cd1-F/Cd1-R, Cd2-F/Cd2-R, Cd1-F/Cd2-R for all methods. It shows the size of the bands (desired gene) for the different samples (strains or isolates).

Mechanical lysis of cells on the different isolates and strains was effective for all methods (Figure 1a (1^st A-C, 3^rd A-C and 5^th A-C) Qiagen + Glass beads; 1b (A, B and C) CTAB + Glass beads; 1c, 1d and 1e ((3^rd A-C), (4th A-C) and (5^th A-C) for Chelex + Glass beads, PCI + Glass beads and CTAB + Glass beads respectively); 1f, 1g and 1h (7 and 10) corresponding to each method for the extract from the reference strain) except for the CTAB + Glass beads method in Figures 1b (3^rd A to C) and 1f, 1g and 1h (4) and CTAB + Sterile sea sand in Figures 1f, 1g and 1h (5). The extracts show a double band for extracts of the 3^rd B and C due to the presence of other species in the extracts and a low band amplitude for extract 7 due to the presence of RNA. Figure 1.c and Figure 1.e show a low amplitude at extract of the 4^th C and Figure 1d show no band for the same extract from the mechanical lysis + PCI method. Thermal lysis shows a weak band with sample 15 with primers Cd1-F_Cd2-R in the Qiagen + Freezing/thawing method (Figure 1a (2^nd A-C, 4^th A-C and 6^th A-C)) due to the presence of RNA. Figure 1.b shows the presence of the gene of interest in samples 2^nd B, 3^rd B, C, and 4^th B and C with the CTAB + Freezing/thawing method with the presence of other species in the samples. Regarding the chemical cell lysis methods, there is an absence of amplification bands for the extracts of the different chemical methods CTAB (1^st A to C) and Chelex + Qiagen ATL lysis buffer (2^nd A to C) in images c, d and e and at the level of extracts 2^nd B and C as shown in images f, g and h in Figure 1 for the same methods. These samples treated with the chemical extraction methods, were not amplified (Figure 1). These results show that extraction methods involving mechanical lysis concentrate DNA better than extraction methods involving thermal lysis and chemical extraction methods.

Figure 2 shows the results of the amplification of extracts with ERG11-ORF primers with the program according to Lee et al. and the optimized program 1 (Cd1_F-Cd1_R). The optimized program 1 amplifies the extracts better than the program according to Lee et al. which amplifies only for one sample (Figure 2). Amplification of extracts (i) according to the program of Lee et al (2004) with the appearance of a band for extract A from the mechanical lysis method combined with Qiagen; no band for extracts B, A and B from the mechanical lysis methods combined with CTAB (B) and thermal lysis methods combined with CTAB (B) and Qiagen (A) respectively. (j) amplification of the extracts according to optimized program 1 with primers Cd1-F_Cd1-R of the extracts from the mechanical lysis methods combined with Qiagen (2^nd A) and CTAB (1^st A) and from the thermal lysis methods combined with Qiagen (2^nd B) and CTAB (1^st B). The same extracts were amplified with ERG11-ORF primers with band formation as for the extracts from Qiagen combined mechanical lysis (2) and Qiagen combined thermal lysis (4).

Amplification with ERG11-ORF primers resulted in bands with program 1 (image k) and with program 2 (image l) only with yeast.

Two types of fungi were used in this study: yeasts and molds. These samples were classified into two groups, the group of isolates (species isolated in the laboratory) which are a yeast of the genus Candida albicans and a mold of the genus Aspergillus fumigatus and a reference strain of the genus ATCC6258 (Table a).

The time required for nucleic acid extraction varied and the protocol duration ranged from 55 minutes for the Chelex ATL method to 4 hours for the CTAB freezing/thawing method (Table 1). Data for the amplification of extracts with the different primers are provided in Table b (supplementary data: mix preparation) and Table d (supplementary data: design of amplification programs).

4. Discussion

Nucleic acid extraction is an essential step in a molecular biology study. The amplification by conventional PCR of the resulting extracts not only makes it possible to assess the quantity and quality of the DNA in the medium but also the characterization of the species involved in the fungal pathology in order to allow its complete identification ³. However, the rigidity of the fungal cell membrane due to its high polysaccharide content requires efficient means to extract gDNA ⁴. Mechanical lysis methods from glass beads, sea sand sterilized and thermally freeze/thawed in this study has shown efficiency in interrupting and isolating the genetic material. Yamada and al. ¹³ showed that the use of mechanical methods by grinding the fungal cell made it possible to break the cell membrane, and Kanshin and al. ¹⁴ showed that the sudden change in extreme temperatures caused the yeast cells to rupture. Post-purification amplicons of these lysates by different purification protocols demonstrates the quality of the extracts from these different lysis methods (Figure 1). Avolio and al, ¹⁵, Rozales and al, ¹⁶ and Tachikawa and al, ¹⁷ showed that amplification of extracts by PCR is an indicator of both quality and sufficient quantities of nucleic acids. This confirms the purity and quality of the different extracts of our protocols. Also, El-Kirat ¹⁸ highlighted the denaturing and inhibitory action of toxic chemicals or buffer such as the Phenol/Chloroform/Isoamyl Alcohol buffer on DNA and PCR, which could be an inhibiter of PCR in our case as shown in Figure 1. Chemicals such as Phenol and Chloroform are hazardous to health. We have not amplified samples with the chemical method because chemical products used to lysate the membrane of the fungal cells had no effect on the fungal cells after purification of the lysates (Figure 1). According to Vingataramin ³, the efficiency of the cell lysis method allows the concentration of DNA which is critical to obtain sufficient DNA for PCR amplification. However, Abdel-Latif & Osman ¹⁹ showed that with the application of a product such as CTAB, it was possible to lyse the fungal cells and obtain a sufficient quantity and a better quality of DNA that could be amplified by PCR. The amplification of the extracts by the different primers confirmed the high quality of DNA products after purification. The sizes of the observed amplification bands indicate the species identity and the accuracy of the primer pairs highlighted here for ITS identification primers (Cd1, Cd2 and Cd1-Cd2). This result partially confirms the identity of the species used in our study. Amplification programs for the identification primers would be suitable for the primers since they allowed the amplification of the extracts.

Amplification of DNA from yeast strains by the ERG11-specific primers of Candida albicans through the amplification program of Lee and al, ²⁰ did not yield any amplification bands (Figure 2 image i). Whereas, amplification of the same DNA products showed amplification products with the optimized program 1 (Figure 2 image K). Similarly, amplification of all extracts from five methods indicated presence of DNA target only in DNA purified from yeast samples (Figure 2 image l). This shows that ERG11-ORF primers are very specific to Candida albicans. Amplifications programs optimized for the amplification of specificity primers would be more suitable for the amplification of these primers. The amplification of the reference strain ATCC6258 confirmed the efficiency of different cell lysis and purification methods. Extraction protocol durations range from 55 minutes to 240 minutes. Dilhari and al, ²¹ have described long protocols ranging from 25 minutes to 11 hours. Long durations of those protocols, costs or toxicity of some products may limit their routine use, but the efficacy of some is worthwhile.

5. Conclusion

Extraction of gDNA from fungal species is difficult and requires the use of sufficiently effective means to break down the cell wall. We have modified, adapted, developed and compared gDNA extraction methods on two groups of fungal species. The best method was based on Chelex combined with glass beads that uses proteinase K because it is less durable and allows extraction of a sufficient amount of gDNA. The lowest cost method was CTAB with sterilized sea sand for cell lysis.

Acknowledgements

We are grateful to John G. Gibbons (University of Massuchetts, US) and David D Denning (University of Mancherster, CEO GAFFI, Global action Fund for Fungal infections) for critical reading and helpful comment of the manuscript. Many thanks to the staff of Molecular Biology Plateform in Pasteur Institute of Cote d’Ivoire for the help and the availability of equipment.

References