DNA extract characterization process for microbial detection methods development and validation

BMC Research Notes

Jayne B Morrow¹  Nathan D Olson¹ 

[1] Biosystems and Biomaterials Division, Material Measurements Laboratory, National Institute of Standard and Technology, 100 Bureau Dr, 20899-8312, Gaithersburg, Maryland

关键词: Biodetection;    qPCR;    PCR inhibition;    Shearing;    DNA purity;    DNA concentration;    DNA characterization;    DNA extraction;   

Others  :  1177964
DOI  :  10.1186/1756-0500-5-668

 received in 2012-07-25, accepted in 2012-11-14,  发布年份 2012

【 摘 要 】

Background

Quantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods.

Results

DNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/μL. Forty-seven extracts had concentrations greater than 17.5 ng/μL, 25 had A₂₆₀/A₂₈₀ above 2.0, and 28 had A₂₆₀/A₂₃₀ ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ μL), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/μL to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/μL and 0.25 ng/μL respectively. The qPCR LOQ varied by cell type (5.72 × 10^-3 ng/μL for E. coli, 2.66 × 10^-3 ng/μL, for B. cereus, 3.78 × 10^-3 ng/μL for B. thailandensis, and 7.67 × 10^-4 ng/μL for S. cerevisiae). A number of samples were below the UV spectroscopy (n = 27), flurometry (n = 15), and qPCR (n = 3) LOQ.

Conclusion

The presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics.

【 授权许可】

   
2012 Olson and Morrow; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JDC, Wengenack NL, Rosenblatt JE, Cockerill FR III, Smith TF: Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006, 19:165-256.
• [2]Rossmanith P, Wagner M: The challenge to quantify Listeria monocytogenes--a model leading to new aspects in molecular biological food pathogen detection. J Appl Microbiol 2011, 110:605-617.
• [3]Holden MJ, Madej RM, Minor P, Kalman LV: Molecular diagnostics: harmonization through reference materials, documentary standards and proficiency testing. Expert Rev Mol Diagn 2011, 11:741-755.
• [4]Janse I, Hamidjaja RA, Bok JM, van Rotterdam BJ: Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification. BMC Microbiol 2010, 10:314.
• [5]Tan SC, Yiap BC: DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol 2009, 2009:574398.
• [6]Kellogg M: Detection of biological agents used for terrorism: are we ready. Clin Chem 2010, 56(1):10-15. Epub 2009 Nov 19
• [7]Wilfinger WW, Mackey K, Chomczynski P: Assessing the quantity, purity and integrity of RNA and DNA following nucleic acid purification. In DNA sequencing II optimizing preparation and cleanup. Edited by Sudbury KJ. MA: Jones and Bartlett Publishers; 2006:291-312.
• [8]Ning J, Liebich J, Kästner M, Zhou J, Schäffer A, Burauel P: Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study. Appl Microbiol Biotechnol 2009, 82:983-993.
• [9]Sambrook J: The condensed protocols from molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2006.
• [10]Glasel JA: Validity of nucleic acids purities monitored by 260 nm/280 nm absorbance ratios. Biotechniques 1994, 18:62-63.
• [11]Simon MC, Gray DI, Cook N, Simon MC, Gray DI: DNA extraction and PCR methods for the detection of Listeria monocytogenes in cold-smoked salmon. Appl Environ Microbiol 1996, 62:822-824.
• [12]Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, Emslie KR: Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 2012, 84:1003-1011.
• [13]Holden MJ, Rabb SA, Tewari YB, Winchester MR: Traceable phosphorus measurements by ICP-OES and HPLC for the quantitation of DNA. Anal Chem 2007, 79:1536-1541.
• [14]Brennan RG, Rabb S, Holden MJ, Winchester MR, Turk GC: Potential primary measurement tool for the quantification of DNA. Anal Chem 2009, 81:3414-3420.
• [15]EURACHEM: The fitness for purpose of analytical methods a laboratory guide to methods validation and related topics. UK: LGC Ltd; 1998.
• [16]Holden MJ, Haynes RJ, Rabb SA, Satija N, Yang K, Blasic JR: Factors affecting quantification of total DNA by UV spectroscopy and picogreen fluorescence. J Agric Food Chem 2009, 57:7221-7226.
• [17]Singer VL, Jones LJ, Yue ST, Haugland RP: Characterization of picogreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal Biochem 1997, 249:228-238.
• [18]Hospodsky D, Yamamoto N, Peccia J: Accuracy, precision, and method detection limits of quantitative PCR for airborne bacteria and fungi. Appl Environ Microbiol 2010, 76:7004-7012.
• [19]Griffiths KR, Burke DG, Emslie KR: Quantitative polymerase chain reaction: a framework for improving the quality of results and estimating uncertainty of measurement. Anal Method 2011, 3:2201.
• [20]Dauphin LA, Hutchins RJ, Bost LA, Bowen MD: Evaluation of automated and manual commercial DNA extraction methods for recovery of Brucella DNA from suspensions and spiked swabs. J Clin Microbiol 2009, 47:3920-3926.
• [21]Dauphin LA, Moser BD, Bowen MD: Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samples. J Microbiol Methods 2009, 76:30-37.
• [22]Dauphin LA, Stephens KW, Eufinger SC, Bowen MD: Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples. J Appl Microbiol 2010, 108:163-172.
• [23]Fahle GA, Fischer SH: Comparison of six commercial DNA extraction kits for recovery of cytomegalovirus DNA from spiked human specimens. J Clin Microbiol 2000, 38:3860-3863.
• [24]Whitehouse CA, Hottel HE: Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples. Mol Cell Probes 2007, 21:92-96.
• [25]Queipo-Ortuño MI, Tena F, Colmenero JD, Morata P, Queipo-Ortuno MI: Comparison of seven commercial DNA extraction kits for the recovery of Brucella DNA from spiked human serum samples using real-time pcr. Eur J Clin Microbiol Infect Dis 2008, 27:109-114.
• [26]Miller DN, Bryant JE, Madsen EL, Ghiorse WC: Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol 1999, 65:4715-4724.
• [27]Swango KL, Timken MD, Chong MD, Buoncristiani MR: A quantitative PCR assay for the assessment of DNA degradation in forensic samples. Forensic Sci Int 2006, 158:14-26.
• [28]Tung N-H, Aboudharam G, Drancourt M: Heat degradation of eukaryotic and bacterial DNA: an experimental model for paleomicrobiology. BMC Res Notes 2012, 5:528. BioMed Central Full Text
• [29]Nolan T, Hands RE, Ogunkolade W, Bustin SA: SPUD: A quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. Anal Biochem 2006, 351:308-310.
• [30]Li R, Mock R, Huang Q, Abad J, Hartung J, Kinard G: A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. J Virol Methods 2008, 154:48-55.
• [31]Kim HS, Byun SH, Lee BM: Effects of chemical carcinogens and physicochemical factors on the UV spectrophotometric determination of DNA. J Toxicol Environ Health A 2005, 68:2081-2095.
• [32]Kushner SR: MRNA decay in prokaryotes and eukaryotes: different approaches to a similar problem. IUBMB Life 2004, 56:585-594.
• [33]Boehm AB, Yamahara KM, Love DC, Peterson BM, McNeill K, Nelson KL: Covariation and photoinactivation of traditional and novel indicator organisms and human viruses at a sewage-impacted marine beach. Environ Sci Technol 2009, 43(21):8046-8052.
• [34]Lim DV, Kearns EA, Leskinen SD, Magana S, Stroot JM, Hunter DM, Schlemmer SM: Sample preparation and assay refinements for pathogen detection platforms. Proc SPIE 2009, 7167:71670O-71670O. 71610
• [35]Courtois S, Frostegård A, Göransson P, Depret G, Jeannin P, Simonet P: Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation. Environ Microbiol 2001, 3:431-439.
• [36]Wilson IG: Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997, 63:3741-3751.
• [37]Mahuku GS: A simple extraction method suitable for PCR - based analysis of fungal and bacterial DNA. Plant Mol Biol Rep 2004, 22:71-81.
• [38]Arbeli Z, Fuentes CL: Improved purification and PCR amplification of DNA from environmental samples. FEMS Microbiol Lett 2007, 272:269-275.
• [39]Pontiroli A, Travis ER, Sweeney FP, Porter D, Gaze WH, Mason S, Hibberd V, Holden J, Courtenay O, Wellington EMH: Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition. PLoS One 2011, 6:e17916.
• [40]Huggett JF, Novak T, Garson JA, Green C, Morris-Jones SD, Miller RF, Zumla A: Differential susceptibility of PCR reactions to inhibitors: an important and unrecognized phenomenon. BMC Res Notes 2008, 1:70. BioMed Central Full Text
• [41]Opel KL, Chung D, McCord BR: A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 2010, 55:25-33.
• [42]Carbonero F, Nava GM, Benefiel AC, Greenberg E, Gaskins RH: Microbial DNA extraction from intestinal biopsies is improved by avoiding mechanical cell disruption. J Microbiol Methods 2012, 87:125-127.
• [43]Mumy KL, Findlay RH: Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR. J Microbiol Methods 2004, 57:259-268.
• [44]Carrigg C, Rice O, Kavanagh S, Collins G, O’Flaherty V: DNA extraction method affects microbial community profiles from soils and sediment. Appl Microbiol Biotechnol 2007, 77:955-964.
• [45]Sauer P, Muller M, Kang J: Quantitation of DNA. Qiagen News 1998, 2:23-26.
• [46]Nolan T, Hands RE, Bustin SA: Quantification of mRNA using real-time rt-PCR. Nat Protoc 2006, 1:1559-1582.
• [47]Demeke T, Jenkins GR: Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem 2010, 396:1977-1990.
• [48]Rothrock MJ Jr: Comparison of microvolume DNA quantification methods for use with volume-sensitive environmental DNA extracts. J Nat and Environ Sci 2011, 2:34-38.
• [49]De Mey M, Lequeux G, Maertens J, De Maeseneire S, Soetaert W, Vandamme E: Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms. Anal Biochem 2006, 353:198-203.
• [50]Nielsen K, Mogensen HS, Hedman J, Niederstätter H, Parson W, Morling N: Comparison of five DNA quantification methods. Forensic Sci Int Genet 2008, 2:226-230.
• [51]Käser M, Ruf M-T, Hauser J, Marsollier L, Pluschke G: Optimized method for preparation of DNA from pathogenic and environmental mycobacteria. Appl Environ Microbiol 2009, 75:414-418.
• [52]Koponen JK, Turunen A-M, Ylä-Herttuala S: Escherichia coli DNA contamination in amplitaq gold polymerase interferes with taqman analysis of lacZ. Mol Ther 2002, 5:220-222.
• [53]Martínez-Blanch JF, Sánchez G, Garay E, Aznar R: Detection and quantification of viable bacillus cereus in food by rt–qPCR. Eur Food Res Technol 2011, 232:951-955.
• [54]Hierro N, Esteve-Zarzoso B, González A, Mas A, Guillamón JM: Real-time quantitative PCR (qPCR) and reverse transcription-qPCR for detection and enumeration of total yeasts in wine. Appl Environ Microbiol 2006, 72:7148-7155.
• [55]Rajendhran J, Gunasekaran P: Strategies for accessing soil metagenome for desired applications. Biotechnol Adv 2008, 26:576-590.
• [56]Purohit MK, Singh SP: Assessment of various methods for extraction of metagenomic DNA from saline habitats of coastal gujarat (India) to explore molecular diversity. Lett Appl Microbiol 2009, 49:338-344.
• [57]Manjula A, Sathyavathi S, Gunasekaran P, Rajendhran J: Comparison of seven methods of DNA extraction from termitarium for functional metagenomic DNA library construction. Ind Res 2011, 70:945-951.
• [58]Da Silva SM, Filliben JJ, Morrow JB: Parameters affecting spore recovery from wipes used in biological surface sampling. Appl Environ Microbiol 2011, 77:2374-2380.
• [59]Sprague LD, Zysk G, Hagen RM, Meyer H, Ellis J, Anuntagool N, Gauthier Y, Neubauer H: A possible pitfall in the identification of burkholderia mallei using molecular identification systems based on the sequence of the flagellin fliC gene. FEMS Immunol Med Microbiol 2002, 34:231-236.
• [60]Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J: Qbase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 2007, 8:R19. BioMed Central Full Text
• [61]Dolezel J, Bartos J, Voglmayr H, Greilhuber J: Nuclear DNA content and genome size of trout and human. Cytometry A 2003, 51:127-128. author reply 129