Application of LATE-PCR to detect Candida and Aspergillus fungal pathogens by a DNA hybridization assay

Dhayaalini Bala Gopal, Chua Ang Lim, Tzar Mohd Nizam Khaithir, Jacinta Santhanam

Research output: Contribution to journalArticle

Abstract

Asymmetric PCR preferentially amplifies one DNA strand for use in DNA hybridization studies. Linear- After-The-Exponential-PCR (LATE-PCR) is an advanced asymmetric PCR method which uses innovatively designed primers at different concentrations. This study aimed to optimise LATE-PCR parameters to produce single-stranded DNA of Candida spp. and Aspergillus spp. for detection via probe hybridisation. The internal transcribed spacer (ITS) region was used to design limiting primer and excess primer for LATEPCR. Primer annealing and melting temperature, difference of melting temperature between limiting and excess primer and concentration of primers were optimized. In order to confirm the presence of singlestranded DNA, the LATE-PCR product was hybridised with digoxigenin labeled complementary oligonucleotide probe specific for each fungal genus and detected using anti-digoxigenin antibody by dot blotting. Important parameters that determine the production of single-stranded DNA in a LATE-PCR reaction are difference of melting temperature between the limiting and excess primer of at least 5°C and primer concentration ratio of excess primer to limiting primer at 20:1. LATE-PCR products of Candida albicans, Candida parapsilosis, Candida tropicalis and Aspergillus terreus at up to 1:100 dilution and after 1 h hybridization time, successfully hybridised to respective oligonucleotide probes with no cross reactivity observed between each fungal genus probe and non-target products. For Aspergillus fumigatus, LATE-PCR products were detected at 1:10 dilution and after overnight hybridisation. These results indicate high detection sensitivity for single-stranded DNA produced by LATE-PCR. In conclusion, this advancement of PCR may be utilised to detect fungal pathogens which can aid the diagnosis of invasive fungal disease.

Original languageEnglish
Pages (from-to)358-364
Number of pages7
JournalMicrobiology and Biotechnology Letters
Volume45
Issue number4
DOIs
Publication statusPublished - 1 Dec 2017

Fingerprint

Aspergillus
Candida
Polymerase Chain Reaction
DNA
Single-Stranded DNA
Freezing
Digoxigenin
Oligonucleotide Probes
Temperature
Candida tropicalis
Aspergillus fumigatus
Mycoses
Candida albicans
Anti-Idiotypic Antibodies

Keywords

  • Fungal pathogens
  • Hybridisation assay
  • LATE-PCR

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Applied Microbiology and Biotechnology

Cite this

Application of LATE-PCR to detect Candida and Aspergillus fungal pathogens by a DNA hybridization assay. / Gopal, Dhayaalini Bala; Lim, Chua Ang; Khaithir, Tzar Mohd Nizam; Santhanam, Jacinta.

In: Microbiology and Biotechnology Letters, Vol. 45, No. 4, 01.12.2017, p. 358-364.

Research output: Contribution to journalArticle

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