Random amplified polymorphic DNA typing of
nosocomial Candida albicans isolates
Somansu Basu, MD,
Harish C. Gugnani, PhD, FRCPath,
Pooja Jain, PhD, Zafar K. Khan, PhD.
population, especially related to HIV infection,
chemotherapy, and organ transplantation.1 Candida
albicans is the most frequent pathogenic species of
candidiasis, and the mortality rate in candidiasis varies
from 38-50%.2,3 Because of its clinical importance,
several typing schemes were developed to assess the strain
identity. These typing methods are generally considered
too variable to be of any practical value in epidemiological
investigations.4 With the advent of molecular genetics,
several DNA-based typing methods are being used
for analyses, such as karyotyping, restriction enzyme
analysis, pulse field gel electrophoresis, and multilocus
enzyme electrophoresis; however, these methods
are laborious and time consuming.5 The analysis of
Candida albicans by random amplified polymorphic
DNA (RAPD) is rapid, convenient, as well as reliable,
and helps better in understanding the epidemiological
aspects of candidiasis.6 In this study, isolates of Candida
albicans from 2 hospitals, Sir Gangaram Hospital and St.
Stephen’s Hospital, in Delhi were analyzed by different
random primers to establish the suitability of the RAPD
typing for Candida strains.
Ten nosocomial isolates of Candida albicans were
recovered from various clinical specimens, such as,
brochoalveolar lavage, blood, urine, pus, and plastic
devices, and confirmed by Analytical Profile Index
Candida. The genomic DNA was extracted according
to the method described by Ausubel with minor
modifications.7 After ribonucleotidase treatment, 2 µl
of extracted DNA template of 50ng/ml concentration
was added into the mixture of 2.5 µl 10X buffer, 0.5 µl
dNTPs mix, 0.2 µl Taq DNA polymerase, 1 µl RAPD
primer, and 18.8 µl standard water to prepare the assay
reaction mixture. Amplification parameters consisted
of 45 cycles of denaturation at 94oC for 60 seconds,
annealing at 35oC for 90 seconds, and extension at
72oC for 90 seconds. In the first cycle, the denaturation
was carried out for 3 minutes, and 10 minutes for the
final extension. Amplification reaction was carried out
in an assay mixture of 25 µl using 6 different RAPD
primers from Operon OPB-11 (5´-GT AG AC CC
GT-3´), OPB 14 (5´-TC CG CT CT GG-3´), OPB 15
(5´-GG AG GG TG TT-3´), OPB 18 (5´-CC AC AG
CA GT-3´), OPB 19 (5´-AC CC CC GA AG-3´), and
OPB 20 (5´-GG AC CC TT AC-3´) in Perkin Elmer
he incidence of candidiasis is dramatically on the
rise due to the increase in the immuno-suppressed
Gene Amplifier 2400. Agarose gel electrophoresis of the
polymerase chain reaction (PCR) product was carried
out in 1.4% gel concentration, with 2 µl of DNA samples
for 2 hours at 2 V/cm electric circuits. The molecular
weight marker used in this study was 1 kb DNA
ladder (Lambda DNA EcoR1Hind III Digest, Sigma).
Genomic DNA with A 260/A 280 ratio in between 1.8
to 2.1 was used, and the RAPD patterns generated was
analyzed using Diversity Database software version 1.1
incorporated into gel documentation system from PDI,
USA. Cluster analysis of the 10 Candida albicans was
carried out using the unweighted–pair group method
with arithmetic average, and the phylogenetic tree was
constructed for all samples in each database.
The application of RAPD technology for strain
delineation of Candida albicans has proven to be a
valuable tool for clinico-epidemiological studies.6,8,9
Gyanchandani et al,10 reported that RAPD of 19 Candida
albicans showed non-identical profile when tested with
21 primers. The present study with oligonucleotide
primers OPB-18, OPB-19, and OPB-20 did not
produce any RAPD profile with the strains. However,
the RAPD patterns of Candida albicans exhibiting
intraspecific polymorphisms were obtained with OPB-
11, OPB-14, and OPB-15 primers. However, unlike
other primers, OPB-14 exhibited RAPD profile with all
the 10 isolates. The OPB-15 showed profiles against 7
isolates only, while OPB-11 showed profiles to 6 isolates
only. The range of molecular weights of DNA obtained
by using OPB-11 was between 1.91 kb and 0.85 kb,
while with OPB-14 it was 728 kb to 278 kb, and with
OPB-15 it was between 1800 bp and 250 bp.
In the last few decades, there has been increasing
reports of Candida infections in India.11 In our study
we found that 3 primers OPB-18, OPB-19, and OPB-
20 were ineffective in producing profiles, but OPB-11
primer when used to generate RAPD profile, 3 clusters
were clearly represented. One band of 1.4 kb was
shared by all the strains when OPB-11 was used. Non-
randomization was found in isolates from blood, urine,
and pus of 3 unrelated patients; and between isolates
of plastic device and skin of 2 unrelated patients in the
same hospital. However, when OPB-15 was used to
amplify the same isolates, it exhibited randomness in
these strains. The OPB-15 primer therefore has better
discriminatory power, with higher detection of strain
variability, and is more efficient in reflecting intra-
Received 16th December 2006. Accepted 17th March 2007.
From the Department of Medical Mycology (Basu, Gugnani), VP Chest Institute,
University of Delhi, and the Department of Mycology (Jain, Khan), CDRI,
Lucknow, India. Current address for correspondence and reprint request to: Dr.
Somansu Basu, Department of Microbiology, Sur Hospital, Ministry of Health,
Oman, PO Box 346, Postal Code 411, Sur, Oman. Tel. +968 25542619. Fax.
+968 25561558. E-mail: email@example.com
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