Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR

Abstract

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood. INTRODUCTIONBlood culture remains the accepted 'gold standard' method for diagnosis of candidiasis and can be highly sensitive. However, in some settings, culture can fail to detect Candida spp. in more than 50 % of patients with chronic disseminated candidiasis MoreiraOliveira et al., 2005). Molecular methods, particularly realtime PCR, are increasingly used alongside conventional microbiological techniques for the diagnosis of systemic candidiasis (Klingspor & Jalal, 2006; Kasai et al., 2006; Löffler et al., 2000;Schabereiter-Gurtner et al., 2007;Bretagne & Costa., 2005;White et al., 2006;Maaroufi et al., 2003; Moreira-Oliveira et al., 2005). However, two major limitations of PCR are the difficulty associated with breaking yeast cell walls to release Candida spp. DNA suitable for amplification (Maaroufi et al., 2004; Löffler et al., 1997) and limited sensitivity when the assays are adapted for testing clinical specimens, such as blood samples, in which the amounts of fungal DNA may be very low (White et al., 2003; Löffler et al., 2000;Ahmad et al., 2002;Challier et al., 2004). As a consequence, these tests are not yet recognized as part of the consensus diagnostic criteria, in contrast to some antigenaemia detection kits (Ascioglu et al., 2002).The yeast cell is notoriously difficult to lyse due to a highly complex cell wall structure that provides rigidity (Müller et al., 1998). Moreover, it has been suggested that the fungal load in blood from ...

Molecular diagnosis of vaginal candidiasis by polymerase chain reaction (PCR) and random amplification polymorphism DNA (RAPD-PCR) in Babylon Province, Iraq

“…The amplicon sizes of this study are same with the results of previous studies that CANIA and CANIB generated amplicon size of 210 bp while CALB1 and CALB2 CALB1 and CALB2 produced amplicon size of approximately 273 bp. These results are in line with that of Luo and Mitchell (2002) and Metwally et al (2008). The PCR results conform to the phenotypic patterns in Figure 1; the specific primers do not amplify the target DNA of non-albican isolates; at the same time, these specific primers amplified the target DNA of C. tropicalis which revealed a blue color.…”

“…was used as described by Metwally et al (2008). This primer pair included CANIA (5'-GAGGGCAAGTCTGGTG-3') and CANIB (5'-CTGCTTTGAACACTCTAA-3').…”

A comparison of molecular methods for hepatitis B virus (HBV) DNA detection from oral fluid samples

“…Both assays are based on silica adsorption, and the QIAamp DNA Mini kit has been used previously for the detection of the hepatitis A virus (HAV) and genomic DNA from oral fluid samples (Mackiewicz et al, 2004;Viltrop et al, 2010), and for fungal (Candida) (Metwally et al, 2008) and cytomegalovirus (CMV) DNA extraction (Evans et al, 1999) from blood samples. To our knowledge, there is no study in which the RTP DNA/RNA Virus Mini kit was used for HBV DNA detection in oral fluid samples.…”

“…In the nucleocapsid, there is an incomplete dsDNA and a DNA polymerase (Seeger & Zoulim, 2007). Real-time and traditional PCR methods are useful for the detection and quantification of infectious agents in clinical specimens (Olive & Bean, 1999;Clarke, 2002;Metwally et al, 2008). These methods can be employed to decide if establishment of HBV treatment is necessary and to monitor the efficacy of that treatment (Dai et al, 2004;Lu et al, 2006).…”

“…Therefore, nucleic acid extraction is an important initial step in molecular diagnostics. Recently, many kits for the extraction of nucleic acids from various types of clinical specimens, such as blood and oral fluid, have been introduced and are commercially available (Durdiaková et al, 2012;Lee et al, 2010;Metwally et al, 2008;Smith et al, 2003;Viltrop et al, 2010).…”