|Year : 2016 | Volume
| Issue : 4 | Page : 533-535
A high yield DNA extraction method for medically important Candida species: A comparison of manual versus QIAcube-based automated system
P Das, P Pandey, A Harishankar, M Chandy, S Bhattacharya
Department of Microbiology, Tata Medical Center, Kolkata, West Bengal, India
|Date of Submission||31-Aug-2015|
|Date of Acceptance||10-Sep-2016|
|Date of Web Publication||8-Dec-2016|
Department of Microbiology, Tata Medical Center, Kolkata, West Bengal
Source of Support: None, Conflict of Interest: None
The prognosis of infected individuals with candidemia depends on rapid and precise diagnosis which enables optimising treatment. Three fungal DNA extraction protocols have been compared in this study for medically important Candida species. The quality and quantity of the DNA extracted by physical, chemical and automated protocols was compared using NanoDrop ND-2000 spectrophotometer. It was found that the yield and purity (260/230) ratio of extracted DNA was significantly high in the physical treatment-based protocol as compared to chemical based or automated protocol. Extracted DNA-based real time-polymerase chain reaction showed an analytical sensitivity of 103 cfu/mL. The result of this study suggests physical treatment is the most successful extraction technique compared to other two protocols.
Keywords: Automated extraction, Candida spp., DNA extraction, manual extraction, real-time polymerase chain reaction
|How to cite this article:|
Das P, Pandey P, Harishankar A, Chandy M, Bhattacharya S. A high yield DNA extraction method for medically important Candida species: A comparison of manual versus QIAcube-based automated system. Indian J Med Microbiol 2016;34:533-5
|How to cite this URL:|
Das P, Pandey P, Harishankar A, Chandy M, Bhattacharya S. A high yield DNA extraction method for medically important Candida species: A comparison of manual versus QIAcube-based automated system. Indian J Med Microbiol [serial online] 2016 [cited 2020 Feb 27];34:533-5. Available from: http://www.ijmm.org/text.asp?2016/34/4/533/195360
| ~ Introduction|| |
Candidemia remains a serious cause of morbidity and mortality worldwide, mainly among immunocompromised and Intensive Care Unit (ICU) patients. For example, candidemia rate of 5.84 per 1000 cancer patient admissions was reported from Taiwan  and 6.51 per 1000 ICU admission was reported from India. Das et al. reported 37% crude mortality from Birmingham UK, MD Anderson Cancer Centre from the USA showed 38% crude mortality and 19% attributable mortality, The molecular diagnosis of candidiasis in recent years have gained in importance because of superior sensitivity, turn-around time. However, universally acceptable protocols for Candida DNA extraction and polymerase chain reaction (PCR) are still lacking.
| ~ Materials and Methods|| |
In this study, an attempt was made for comparison of three DNA extraction procedures based on a single commercial DNA extraction kit (Qiagen, USA) for the most clinically important Candida species and finally verify the quality of the DNA by a Candida real-time PCR based assay. Five reference yeast strains of Candida albicans ATCC 90028, Candida tropicalis ATCC 66029, Candida glabrata ATCC MYA 2950, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258 and three clinical isolates Candida famata, Candida dubliniensis, Candida haemulonii [Figure 1] was used for this study. All the DNA extraction protocols were initially started from a pure culture. Colonies were cultured on Sabouraud dextrose agar plates after incubation for 24–48 h at 37°C. Enumeration of the Candida suspensions was cross-checked using a haemocytometer. Three different protocols were attempted from this step onwards.
Protocol A: Using QIAamp DNA mini kit, with prior chemical treatment. In this protocol, we introduced a chemical treatment with sodium dodecyl sulphate and/or β-mercaptoethanol, before commercial kit extraction step.
Protocol B: QIAamp DNA mini kit with glass beads. Extraction was performed using a modification of protocol A, where the chemical treatment step was replaced with boiling for 10 min followed by freezing the sample at −50°C and agitation with 0.5 mm glass beads. Qiagen buffer ATL was added to each tube and a volume of sterile glass beads approximately equal to 100 µl was added. Samples were processed for 30 s pulse at 5000 rpm in a Mini-BeadBeater, followed by centrifugation at 13,200 rpm in a centrifuge for 10 min.
Protocol C: This protocol used completely automated QIAcube extraction using QIAamp DNA mini kit. For this protocol, the initial concentration of the sample was fixed 0.5–1.0 McFarland standard.
All tested protocols were repeated at least three times for reproducibility of yield and quality. The integrity of the genomic DNA was checked by three different methods. First, NanoDrop 2000 spectrophotometer in triplicate and the results to determine the DNA concentration. In addition, the absorbance at 260 nm (A260) was measured and the A260/A280 ratio determined for the purity of DNA. Second, each extracted DNA was visually checked under UV-light after electrophoresis on 0.8% agarose gels, using 100 bp reference marker. Finally, the yield and purity of extracted DNA was verified by Real time PCR assay targeting rRNA gene. The TaqMan-based Real time PCR assay was carried out with the custom designed rRNA region specific primers and probes. Specificity of the primers and probes were checked using NCBI blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast).
| ~ Results|| |
Within each protocol, major differences in yield and purity were observed between the three individual tested protocols from the same amount of initial inoculum concentrations used (0.6–0.8 OD at 600 nm standard). Protocol B achieved the best result. All the samples had DNA concentration in the range from 100 to 198 ± 18.9 ng/µl (tested in triplicate), and the purity was 1.7–2.1 ± 0.8.
Protocol A had just 50% of samples yielding not more than 20 ng/µl of DNA, and their purity was 1.5–2.0 ± 0.4. QIAcube extraction (Protocol C) showed a poor DNA yield compared to other tested protocols [Figure 1].
To check the purity and yield of the three different DNA extraction protocol, we have used a PAN Candida Real time PCR, targeting 18S rRNA region. The DNA extracted from protocol A and C were of poor quality and not reproducibly amplifiable by PCR. Whereas the DNA extract obtained by protocol B was consistently amplifiable.
The Candida PCR showed an analytical sensitivity of 103 cfu/mL [Table 1]. Four Candida species, namely, C. glabrata, C. krusei, C. haemulonii and C. dubliniensis, did not amplify by the present primers. The analytical specificity of the Candida PCR was checked against 13 different DNA samples from bacteria, virus and other fungus and was found to be specific. The specificity and sensitivity of the Candida PCR for the same four Candida sp. were also checked by human blood spiking experiments and it gave similar results.
|Table 1: The analytical sensitivity of the Pan Candida sp. specific real-time polymerase chain reaction for four medically important Candida sp. using the DNA extraction protocol B|
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| ~ Discussion|| |
In the present study, we described and validated a new format for DNA extraction method for medically important Candida sp. This study also showed a significant difference in terms of yield and purity of the extracted DNA by different protocols. Our data also support the possibility that impurities during extraction may act as inhibitors for PCR assays  as seen for the DNA extracts of protocol A and C. The analytical sensitivity of the Candida Real time PCR using DNA extracted by protocol B proves the integrity of DNA obtained. Many studies from India and abroad showed more than 90% of the invasive infections due to Candida are attributed to five species – C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei., A recently published data from our hospital also showed Candida to be responsible for 6% of all bloodstream infections. C. albicans to be the most common species in our centre but C. glabrata, C. haemulonii, C. kefyr, C. norvegensis, C. parapsilosis and C. tropicalis were also noted. Based on our microbiological data, an attempt was made for the development of PCR for rapid identification of those Candida sp. which are commonly found in our setup. A number of molecular-based detection strategies (e.g., sequence-based analysis) are used for identification of the Candida currently used in the clinical microbiology laboratories. However, many of them are both expensive and laborious or time-consuming.
| ~ Conclusions|| |
From the clinical perspectives, the establishment of simple, inexpensive DNA extraction technique and PCR assay that are readily adaptable to the medical microbiology laboratory remains an important clinical and technical requirement which our assay has attempted to address.
Financial support and sponsorship
The study was supported by the Department of Biotechnology, Government of India. No.BT/PR4884/MED/29/394/2012.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Blot S, Dimopoulos G, Rello J, Vogelaers D. Is Candida
really a threat in the ICU? Curr Opin Crit Care 2008;14:600-4.
Tang HJ, Liu WL, Lin HL, Lai CC. Epidemiology and prognostic factors of candidemia in cancer patients. PLoS One 2014;9:e99103.
Chakrabarti A, Sood P, Rudramurthy SM, Chen S, Kaur H, Capoor M, et al.
Incidence, characteristics and outcome of ICU-acquired candidemia in India. Intensive Care Med 2015;41:285-95.
Das I, Nightingale P, Patel M, Jumaa P. Epidemiology, clinical characteristics, and outcome of candidemia: Experience in a tertiary referral center in the UK. Int J Infect Dis 2011;15:e759-63.
Sipsas NV, Lewis RE, Tarrand J, Hachem R, Rolston KV, Raad II, et al.
Candidemia in patients with hematologic malignancies in the era of new antifungal agents (2001-2007): Stable incidence but changing epidemiology of a still frequently lethal infection. Cancer 2009;115:4745-52.
Avni T, Leibovici L, Paul M. PCR diagnosis of invasive candidiasis: Systematic review and meta-analysis. J Clin Microbiol 2011;49:665-70.
Katcher HL, Schwartz I. A distinctive property of Tth DNA polymerase: Enzymatic amplification in the presence of phenol. Biotechniques 1994;16:84-92.
Giri S, Kindo AJ, Kalyani J. Candidemia in intensive care unit patients: A one year study from a tertiary care center in South India. J Postgrad Med 2013;59:190-5.
Khan PA, Fatima N, Nabeela, Jahan S, Khan HM, Malik A. Antifungal susceptibility pattern of Candida
isolates from a tertiary care hospital of North India: A five year study. Int J Curr Microbiol Appl Sci 2015;1:177-81.
Bhattacharya S, Goel G, Mukherjee S, Bhaumik J, Chandy M. Epidemiology of antimicrobial resistance in an oncology center in Eastern India. Infect Control Hosp Epidemiol 2015;36:864-6.
Boyanton BL Jr., Luna RA, Fasciano LR, Menne KG, Versalovic J. DNA pyrosequencing-based identification of pathogenic Candida
species by using the internal transcribed spacer 2 region. Arch Pathol Lab Med 2008;132:667-74.
Putignani L, Paglia MG, Bordi E, Nebuloso E, Pucillo LP, Visca P. Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25-28S rRNA gene. Mycoses 2008;51:209-27.