NEW MICROBIOLOGICA, 29, 111-119, 2006 Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples Massimiliano Bergallo, Cristina Costa, Giorgio Gribaudo, Sonia Tarallo, Sara Baro, Alessandro Negro Ponzi, Rossana Cavallo Department of Public Health and Microbiology, Virology Unit, University of Turin, Italy SUMMARY The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors. KEY WORDS: PCR, DNA extraction, BKV, PCR inhibitors Received February 8, 2006 Accepted March 14, 2006 INTRODUCTION sequences has allowed the determination of pres- ence and quantification of specific viral genes and Molecular biology techniques represent a pow- sequences in biological matrices. Indeed, the erful tool for nucleic acid analysis, enabling the detection of nucleic acid sequences from many detection of even a single genomic copy and are viruses, including polyomavirus BK, in serum widely used for diagnostic and research purposes. and urine samples for routine diagnostics is cur- The target nucleic acid sequences may be rently performed by PCR in most laboratories. amplified by PCR and the product (amplicon) The success and reliability of nucleic acid detected in ethidium bromide-stained gel or by sequences amplification require efficient unbi- colorimetric enzyme immunoassay. In particu- ased procedures of extraction and purification lar, the ability of PCR to amplify nucleic acid from other microrganism components, biolog- ical substances, and contaminating materials which could function as PCR inhibitors Corresponding author (Wiedbrauk et al., 1995). An efficient unbiased Rossana Cavallo method of DNA extraction that yields pure, high- Department of Public Health and Microbiology, quality DNA is crucial for the success of PCR and Virology Unit University of Turin, Via Santena, 9 sequencing reactions and the subsequent treat- 10126 Torino, Italy ment of disease (McOrist et al., 2002). One of the E-mail: address: [email protected] major limitations of PCR-based tests is the inhi- 112 M. Bergallo, C. Costa, G. Gribaudo, S. Tarallo, S. Baro, A. Negro Ponzi, R. Cavallo bition of the nucleic acid amplification process traditionally used to obtain nucleic acids from by substances present in clinical samples. For cells and microorganisms. In addition to sepa- example, DNAse or RNAse can degrade target ration and purification from contaminating mate- nucleic acid and/or oligonucleotide primers rials, this method minimizes the inhibition of (Mullen et al., 1990); EDTA can chelate divalent nucleic acid amplification by PCR in clinical sam- cations such as Mg2+ that are essential for the ples (Khan et al., 1991; Ruano et al., 1992; activity of the thermostable enzyme Taq poly- Wiedbrauk et al., 1995). The aim of this study was merase (Greenfield et al., 1993); different mate- to compare six different DNA extraction proce- rials such as heparin (Holodniy et al., 1991; Izraeli dures from urine and serum samples for subse- et al., 1991), phenol (Katcher et al., 1994), dena- quent PCR analysis. Using specimens spiked with tured albumin (Vandenvelde et al., 1993), known amounts of BK virus DNA, the following polyamines (Ahokas et al., 1993), polysaccharides extraction protocols were compared: a com- (Shioda et al., 1987), and calcium alginate mercial Kit (Extragen), a lysis extraction pro- (Wadowsky et al., 1994) inhibit PCR by direct cedure which uses a lysis buffer, a lysis extrac- effect on Taq polymerase. Haem, the prosthetic tion procedure which employs a lysis buffer in group of hemoglobin released from erythrocytes conjunction with a hot pretreatment, a phe- following hemolysis and frequently associated nol/chloroform extraction followed by ethanol with blood sampling, reversibly binds to Taq poly- precipitation, a lysis extraction which utilizes a merase inhibiting its activity (Byrnes et al., 1975; guanidium thiocyanate/silica matrix method, and Levere et al., 1991; Mercier et al., 1990; Tsutsui a lysis extraction procedure which utilizes a lysis and Mueller, 1987). As little as 1% (vol/vol) blood buffer in conjunction with proteinase K. The six completely inhibits Taq polymerase activity extraction protocols were evaluated on the (Panaccio and Lew, 1991). Urine has been found basis of the following criteria: total yield of DNA, to be a particularly difficult specimen for PCR- purity of the DNA recovered, and presence of based amplification, due to the presence of many PCR-inhibitory substances. In addition, physical, possible inhibitors (Demmler et al., 1988; chemical, and enzymatic elements were briefly Yamaguchi et al. 1992). For example, concen- analyzed. trations of 20 mM urea and above are inhibito- ry for PCR (Khan et al., 1991). Several studies have been devoted to the development of sam- MATERIALS AND METHODS ple pretreatments to obtain adequate specimens (Lantz et al., 2000) or to enhance the efficiency Experimental design of PCR using an alternative thermostable DNA In order to evaluate DNA extraction and purifi- polymerase more resistant to inhibitors (Abu Al- cation procedures, a known amount of BK virus Soud and Rådström, 1998; Katcher and DNA was added to biological samples and the Schwartz, 1994; Panaccio and Lew, 1991; amount recovered after extraction and purifi- Wiedbrauk et al., 1995) or amplification facili- cation was determined. Serum and urine spec- tators such as bovine serum albumin, the single- imens were obtained from 10 healthy volunteers stranded DNA binding T4 gene 32 protein (gp32), and stored at -30°C. All specimens were previ- organic solvents, and proteinase inhibitors (Abu ously tested for BK virus DNA by nested PCR and Al-Soud and Rådström, 2000; Akane et al., 1994; resulted negative. Chandler et al., 1998; de Lomas et al., 1992; BK virus DNA-negative sera and urines were Demeke and Adams, 1992; Gilgen et al., 1995; spiked with a known amount (400 ng) of plas- Izraeli et al., 1991; Kreader, 1996; Pomp and mid pBKV 33-1 (obtained from the American Medrano, 1991; Powell et al., 1994; Vandenvelde Type Culture Collection [Rockeville, USA]), con- et al., 1993). Many lysis and DNA extraction meth- taining the whole sequence of the polyomavirus ods have been described in literature BK. The spiked samples were then extracted by (Behzadbehbahani et al., 1997; Bowtell, 1987; the six different extraction methods and tested Ciulla et al., 1988). The phenol-chloroform by semiquantitative PCR. Extraction proce- extraction procedure for DNA (Sambrook et al., dures were performed for each specimen in quin- 1989) followed by ethanol precipitation has been tuplicate, giving a total number of 50 extractions Evaluation of methods for DNA extraction 113 from urine samples and 50 extractions from 10 min, and then centrifuged for 15 min at 13,000 serum samples. Evaluation of extraction proto- rpm at room temperature. A total of 250 µl of cols was based on three features: supernatant of boiled samples was added to 250 1) recovery efficiency in terms of yield of DNA µl of lysis buffer (400 mM Tris-HCl pH 7.5, 500 and percentage of plasmid pBKV recovered mM NaCl, 50 mM EDTA, 1% SDS), incubated in each sample, as determined using a spec- for 30 min, then centrifuged for 5 min at 13,000 trophotometer; rpm at room temperature. Four hundred µl of 2) purity of extracted DNA based on spec- the supernatant were mixed with 400 µl of iso- trophotometric readings of the 260/280 nm- propanol and incubated at -30°C for 15 min. After absorbance ratio (pure DNA has a 260/280 centrifugation for 5 min at 13,000 rpm at room nm-absorbance ratio equal to 1.8); temperature, the pellet was washed with 70% 3) presence of inhibitors, as estimated by a PCR ethanol, centrifuged for 5 min at 13,000 rpm at inhibition assay in which 1 ng of extracted room temperature, air-dried for at least 30 min, DNA were used as template for semiquanti- resuspended in 20 µl of double-distilled H2O, and tative PCR amplification of BK virus Large stored at -20°C prior to use. T antigen. One target-free control for every sample was Protocol C: home-made extraction procedure #2. included in each test run. In order to reduce the A total of 250 µl of each sample was added to likelihood of contamination, specimen extraction 250 µl of lysis buffer (400 mM Tris-HCl pH 7.5, and analysis of amplified products were per- 500 mM NaCl, 50 mM EDTA, 1% SDS), incubated formed in separate rooms (Espy