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Comparison of manual and automated nucleic acid extraction methods from clinical specimens for microbial diagnosis purposes
Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Note
Comparison of manual and automated nucleic acid extraction methods from clinical specimens for microbial diagnosis purposes Aniela Wozniak ⁎, Enrique Geoffroy, Carolina Miranda, Claudia Castillo, Francia Sanhueza, Patricia García Laboratorio de Microbiología, Departamento de Laboratorios Clínicos, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
a r t i c l e
i n f o
Article history: Received 14 April 2016 Received in revised form 21 June 2016 Accepted 8 July 2016 Available online xxxx Keywords: MagNA Pure Molecular diagnosis Nucleic acid extraction methods Microbial pathogens
a b s t r a c t The choice of nucleic acids (NAs) extraction method for molecular diagnosis in microbiology is of major importance because of the low microbial load, different nature of microorganisms, and clinical specimens. The NA yield of different extraction methods has been mostly studied using spiked samples. However, information from real human clinical specimens is scarce. The purpose of this study was to compare the performance of a manual low-cost extraction method (Qiagen kit or salting-out extraction method) with the automated high-cost MagNAPure Compact method. According to cycle threshold values for different pathogens, MagNAPure is as efficient as Qiagen for NA extraction from noncomplex clinical specimens (nasopharyngeal swab, skin swab, plasma, respiratory specimens). In contrast, according to cycle threshold values for RNAseP, MagNAPure method may not be an appropriate method for NA extraction from blood. We believe that MagNAPure versatility reduced risk of cross-contamination and reduced hands-on time compensates its high cost. © 2016 Elsevier Inc. All rights reserved.
The yield and quality of nucleic acids (NAs) are crucial for the performance of molecular tests for microbial detection (Muldrew, 2009; Ince & McNally, 2009; Burd, 2010). Manual extraction kits have demonstrated good performance but are too laborious and have a high risk of crosscontamination (Yang et al., 2008). Automated extraction methods like MagNA Pure System (Roche) (MP) provide fast and high-throughput extraction capacity (Akutsu et al., 2004). Extraction of human DNA from clinical samples for genetic diagnosis is efficient due to a high number of human cells in the sample material. However, microbial load in clinical samples is often very low. The NA yield of different extraction methods has been mostly studied using spiked samples (Goldschmidt, 2014; Yera et al., 2009; Cowart & Winchell, 2009; L. et al., 2013; Edvinsson et al., 2004). In contrast, there is much less information about NA extraction from actual clinical specimens. The purpose of this study was to compare the performance of manual extraction methods QIAamp DNAMinikit (QIAGEN) (Q) and a salting-out extraction method (Lahiri & Nurnberger, 1991) with MP for NA extraction from clinical specimens for microbial diagnosis purposes. We analyzed the efficiency of NA extraction through the analysis of cycle threshold (CT) values for each specific pathogen and for an internal quality control (human RNAseP gene, and a synthetic internal control for plasma samples). We included 21 nasopharyngeal swabs for the diagnosis of Bordetella pertussis (BP) (7 positive, 14 negative), 20 skin swabs for Herpes simplex ⁎ Corresponding author at: Laboratorio de Microbiología, Pontificia Universidad Católica de Chile, Centro Médico San Joaquín, Vicuña Mackenna 4686, 3rd floor, Santiago 7820436, Chile Tel.: +562-23548573; Fax: +562-23548571. E-mail addresses: [email protected], [email protected] (A. Wozniak).
1/2 virus (HSV) (9 positive, 11 negative), 23 respiratory specimens for Pneumocystis jirovecii (PJ) (7 positive, 16 negative), 20 plasma samples for Hepatitis C virus (HCV) (10 positive, 10 negative), and 76 whole blood samples for Tripanosoma cruzi or Toxoplasma gondii. Samples for detection of BP, PJ, and HSV were extracted in parallel through both methods Q and MP following manufacturer instructions. Plasma samples for HCV were extracted using QIAamp viral RNAMinikit (QIAGEN) according to manufacturer instructions. Of the 76 blood samples, 38 were extracted through a salting-out manual method (Lahiri & Nurnberger, 1991), and 38 with MP. DNAs extracted from blood were analyzed through quantitative polymerase chain reaction for RNAseP gene only. Detection of all pathogens was done through quantitative polymerase chain reaction using commercial kits or in-house-developed methods. Detection of HSV was made with HSV 1/2 Detection Kit (Roche) according to manufacturer instructions. Detection of PJ was made as described and informed positive when CT was below 39 (Larsen et al., 2002). Amplification of HSV and PJ were performed in a LightCycler 2.0 real-timepolymerase chain reaction (PCR) equipment (Roche). Detection of BP was made as described and informed positive when CT was below 36 (Kosters et al., 2001). Detection of HCV was performed using COBAS Taqman HCV Test v2.0 according to manufacturer instructions. Amplification of HCV and BP were performed in a StepOne Real-Time PCR equipment (Applied Biosystems, Foster City, CA, United States). Amplification of RNAseP was made as described and the internal control was considered validated if CT value was below 31 (Luo et al., 2005). It was observed that the average CT values for HCV, BP, and PJ were similar for samples extracted with Q and with MP (Table 1A), whereas values for HSV obtained with MP were significantly lower than with
http://dx.doi.org/10.1016/j.diagmicrobio.2016.07.008 0732-8893/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Wozniak A, et al, Comparison of manual and automated nucleic acid extraction methods from clinical specimens for microbial diagnosis purposes, Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.07.008
2
A. Wozniak et al. / Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx
Table 1 CT values for HSV, BP, HCV, and PJ (A), and CT values obtained for internal control amplification (B). Qiagen A HSV BP HCV PJ B Skin swab (HSV) Nasopharyngeal swab (BP) Plasma (HCV)⁎ Respiratory specimens (PJ)
MagNA Pure
Student's t test§
Avg Δ CT ([Q-MP] ± SE)
Avg specific target (CT ± SE) 23.77 ± 1.62 20.82 ± 2.22 26.01 ± 0.69 30.81 ± 2.63
22.53 ± 1.57 20.74 ± 2.29 26.31 ± 0.81 29.85 ± 2.66
P = 0.007 NS NS NS
1.24 ± 0.34 0.08 ± 0.52 −0.30 ± 0.46 0.96 ± 0.42
Avg internal control⁎ (CT ± SE) 29.43 ± 0.91 26.07 ± 0.30 29.86 ± 0.75 27.04 ± 0.58
28.94 ± 0.95 25.00 ± 0.30 29.26 ± 0.61 26.58 ± 0.58
NS P = 0.0007 NS NS
0.49 ± 0.25 1.07 ± 0.26 0.59 ± 0.34 0.46 ± 0.30
CT = cycle threshold; Avg = average; NS = not significant. § Student's t test performed between values of CTs from specimens extracted with Q and MP. ⁎ Internal control was human RNAseP gene except in plasma samples in which a synthetic plasmid was used.
Q. The difference between CT values obtained through both methods for each sample was positive for HSV, BP, and PJ (Table 1A). In contrast, the difference was negative for HCV, maybe because inefficient RNA extraction as reported previously (Yang et al., 2011). CT values obtained for RNAseP from skin and nasopharyngeal swabs, respiratory specimens, and plasma extracted with Q were higher than with MP (Table 1B). The average CT difference calculated for each sample was positive for all sample types (Table 1B). We believe MP is a fast and effective method for NA extraction from nasopharyngeal and skin swabs, respiratory specimens and plasma. In contrast, the CT values for RNAseP were 5 cycles higher for blood samples extracted through MP compared with the salting-out method (Fig. 1B). Because hemoglobin present in blood inhibits PCR (Bessetti, 2007), we repurified MP-extracted DNAs with QIAGEN silica-column. However, CT values remained high (Fig. 1), maybe because extraction efficiency is low in blood specimens. We believe MP is not an appropriate method for NA extraction from blood for microbial diagnosis purposes. The choice of NA extraction method is a factor of major importance because it can impact diagnostic accuracy. In our location, the cost of MP per extraction is approximately 50% higher than Q. However, hands-on time is much higher for Q. We consider MP a highly convenient method for NA extraction of different kinds of pathogens from
NS
CT RNAse P
30
***
20
10
0 salting-out
MP
MP + Q
Fig. 1. CT values for human RNAseP amplification from blood samples extracted with the salting-out manual method, MP, and MP followed by purification with QIAGEN silica column (MP + Q). ***P b 0.0001, unpaired Student's t test between values of CTs from samples extracted with salting-out method and MP. NS, non-significant, paired Student's t test between values of CTs from samples extracted with MP and with MP + Q.
noncomplex specimens: its versatility, reduced risk of crosscontamination, and reduced hands-on time compensates its high cost. Acknowledgments This work was supported by research funds from the Department of Clinical Laboratories at Pontificia Universidad Católica de Chile. We acknowledge the personnel of Laboratorio de Microbiología, Pontificia Universidad Católica de Chile, for help with technical aspects of this work. The authors of this article declare they have no conflicts of interest. References Akutsu J, Tojo Y, Segawa O, Obata K, Okochi M, Tajima H, et al. Development of an integrated automation system with a magnetic bead-mediated nucleic acid purification device for genetic analysis and gene manipulation. Biotechnol Bioeng 2004;86: 667–71. Bessetti J. An introduction to PCR inhibitors. Profiles DNA 2007;10:9–10. Burd EM. Validation of laboratory-developed molecular assays for infectious diseases. Clin Microbiol Rev 2010;23:550–76. A. TK, Cowart KC, Winchell JM. Comparison-of-nucleic-acid-extraction-methods-for-thedetection-of-mycoplasma-pneumoniae. Diagn Microbiol Infect Dis 2009;65:435–8. Edvinsson B, Jalal S, Nord CE, Pedersen BS, Evengard B. DNA extraction and PCR assays for detection of Toxoplasma gondii. Apmis 2004;112:342–8. Goldschmidt P. S. Degorge, L. Merabet, and C. Chaumeil, 'Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious Agents'. PLoS One 2014;9, e94886. Ince J, McNally A. Development of rapid, automated diagnostics for infectious disease: advances and challenges. Expert Rev Med Devices 2009;6:641–51. Kosters K, Riffelmann M, Wirsing von Konig CH. Evaluation of a real-time PCR assay for detection of Bordetella pertussis and B. Parapertussis in clinical samples. J Med Microbiol 2001;50:436–40. L. PN, Elrod MG, Newton BR, Dauphin LA, Shi J, Chawalchitiporn S, et al. Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR. PLoS One 2013;8:e58032. Lahiri DK, Nurnberger Jr JI. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 1991;19:5444. Larsen HH, Masur H, Kovacs JA, Gill VJ, Silcott VA, Kogulan P, et al. Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia. J Clin Microbiol 2002;40:490–4. Luo W, Yang H, Rathbun K, Pau C-P, Chin-Yih O. Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay. J Clin Microbiol 2005;43:1851–7. Muldrew KL. Molecular diagnostics of infectious diseases. Curr Opin Pediatr 2009;21: 102–11. Yang JL, Wang MS, Cheng AC, Pan KC, Li CF, Deng SX. A simple and rapid method for extracting bacterial DNA from intestinal microflora for ERIC-PCR detection. World J Gastroenterol 2008;14:2872–6. Yang G, Erdman DE, Kodani M, Kools J, Bowen MD, Fields BS. Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens. J Virol Methods 2011;171:195–9. Yera H, Filisetti D, Bastien P, Ancelle T, Thulliez P, Delhaes L. Multicenter comparative evaluation of five commercial methods for toxoplasma DNA extraction from amniotic fluid. J Clin Microbiol 2009;47:3881–6.
Please cite this article as: Wozniak A, et al, Comparison of manual and automated nucleic acid extraction methods from clinical specimens for microbial diagnosis purposes, Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.07.008
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Note
Comparison of manual and automated nucleic acid extraction methods from clinical specimens for microbial diagnosis purposes Aniela Wozniak ⁎, Enrique Geoffroy, Carolina Miranda, Claudia Castillo, Francia Sanhueza, Patricia García Laboratorio de Microbiología, Departamento de Laboratorios Clínicos, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
a r t i c l e
i n f o
Article history: Received 14 April 2016 Received in revised form 21 June 2016 Accepted 8 July 2016 Available online xxxx Keywords: MagNA Pure Molecular diagnosis Nucleic acid extraction methods Microbial pathogens
a b s t r a c t The choice of nucleic acids (NAs) extraction method for molecular diagnosis in microbiology is of major importance because of the low microbial load, different nature of microorganisms, and clinical specimens. The NA yield of different extraction methods has been mostly studied using spiked samples. However, information from real human clinical specimens is scarce. The purpose of this study was to compare the performance of a manual low-cost extraction method (Qiagen kit or salting-out extraction method) with the automated high-cost MagNAPure Compact method. According to cycle threshold values for different pathogens, MagNAPure is as efficient as Qiagen for NA extraction from noncomplex clinical specimens (nasopharyngeal swab, skin swab, plasma, respiratory specimens). In contrast, according to cycle threshold values for RNAseP, MagNAPure method may not be an appropriate method for NA extraction from blood. We believe that MagNAPure versatility reduced risk of cross-contamination and reduced hands-on time compensates its high cost. © 2016 Elsevier Inc. All rights reserved.
The yield and quality of nucleic acids (NAs) are crucial for the performance of molecular tests for microbial detection (Muldrew, 2009; Ince & McNally, 2009; Burd, 2010). Manual extraction kits have demonstrated good performance but are too laborious and have a high risk of crosscontamination (Yang et al., 2008). Automated extraction methods like MagNA Pure System (Roche) (MP) provide fast and high-throughput extraction capacity (Akutsu et al., 2004). Extraction of human DNA from clinical samples for genetic diagnosis is efficient due to a high number of human cells in the sample material. However, microbial load in clinical samples is often very low. The NA yield of different extraction methods has been mostly studied using spiked samples (Goldschmidt, 2014; Yera et al., 2009; Cowart & Winchell, 2009; L. et al., 2013; Edvinsson et al., 2004). In contrast, there is much less information about NA extraction from actual clinical specimens. The purpose of this study was to compare the performance of manual extraction methods QIAamp DNAMinikit (QIAGEN) (Q) and a salting-out extraction method (Lahiri & Nurnberger, 1991) with MP for NA extraction from clinical specimens for microbial diagnosis purposes. We analyzed the efficiency of NA extraction through the analysis of cycle threshold (CT) values for each specific pathogen and for an internal quality control (human RNAseP gene, and a synthetic internal control for plasma samples). We included 21 nasopharyngeal swabs for the diagnosis of Bordetella pertussis (BP) (7 positive, 14 negative), 20 skin swabs for Herpes simplex ⁎ Corresponding author at: Laboratorio de Microbiología, Pontificia Universidad Católica de Chile, Centro Médico San Joaquín, Vicuña Mackenna 4686, 3rd floor, Santiago 7820436, Chile Tel.: +562-23548573; Fax: +562-23548571. E-mail addresses: [email protected], [email protected] (A. Wozniak).
1/2 virus (HSV) (9 positive, 11 negative), 23 respiratory specimens for Pneumocystis jirovecii (PJ) (7 positive, 16 negative), 20 plasma samples for Hepatitis C virus (HCV) (10 positive, 10 negative), and 76 whole blood samples for Tripanosoma cruzi or Toxoplasma gondii. Samples for detection of BP, PJ, and HSV were extracted in parallel through both methods Q and MP following manufacturer instructions. Plasma samples for HCV were extracted using QIAamp viral RNAMinikit (QIAGEN) according to manufacturer instructions. Of the 76 blood samples, 38 were extracted through a salting-out manual method (Lahiri & Nurnberger, 1991), and 38 with MP. DNAs extracted from blood were analyzed through quantitative polymerase chain reaction for RNAseP gene only. Detection of all pathogens was done through quantitative polymerase chain reaction using commercial kits or in-house-developed methods. Detection of HSV was made with HSV 1/2 Detection Kit (Roche) according to manufacturer instructions. Detection of PJ was made as described and informed positive when CT was below 39 (Larsen et al., 2002). Amplification of HSV and PJ were performed in a LightCycler 2.0 real-timepolymerase chain reaction (PCR) equipment (Roche). Detection of BP was made as described and informed positive when CT was below 36 (Kosters et al., 2001). Detection of HCV was performed using COBAS Taqman HCV Test v2.0 according to manufacturer instructions. Amplification of HCV and BP were performed in a StepOne Real-Time PCR equipment (Applied Biosystems, Foster City, CA, United States). Amplification of RNAseP was made as described and the internal control was considered validated if CT value was below 31 (Luo et al., 2005). It was observed that the average CT values for HCV, BP, and PJ were similar for samples extracted with Q and with MP (Table 1A), whereas values for HSV obtained with MP were significantly lower than with
http://dx.doi.org/10.1016/j.diagmicrobio.2016.07.008 0732-8893/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Wozniak A, et al, Comparison of manual and automated nucleic acid extraction methods from clinical specimens for microbial diagnosis purposes, Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.07.008
2
A. Wozniak et al. / Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx
Table 1 CT values for HSV, BP, HCV, and PJ (A), and CT values obtained for internal control amplification (B). Qiagen A HSV BP HCV PJ B Skin swab (HSV) Nasopharyngeal swab (BP) Plasma (HCV)⁎ Respiratory specimens (PJ)
MagNA Pure
Student's t test§
Avg Δ CT ([Q-MP] ± SE)
Avg specific target (CT ± SE) 23.77 ± 1.62 20.82 ± 2.22 26.01 ± 0.69 30.81 ± 2.63
22.53 ± 1.57 20.74 ± 2.29 26.31 ± 0.81 29.85 ± 2.66
P = 0.007 NS NS NS
1.24 ± 0.34 0.08 ± 0.52 −0.30 ± 0.46 0.96 ± 0.42
Avg internal control⁎ (CT ± SE) 29.43 ± 0.91 26.07 ± 0.30 29.86 ± 0.75 27.04 ± 0.58
28.94 ± 0.95 25.00 ± 0.30 29.26 ± 0.61 26.58 ± 0.58
NS P = 0.0007 NS NS
0.49 ± 0.25 1.07 ± 0.26 0.59 ± 0.34 0.46 ± 0.30
CT = cycle threshold; Avg = average; NS = not significant. § Student's t test performed between values of CTs from specimens extracted with Q and MP. ⁎ Internal control was human RNAseP gene except in plasma samples in which a synthetic plasmid was used.
Q. The difference between CT values obtained through both methods for each sample was positive for HSV, BP, and PJ (Table 1A). In contrast, the difference was negative for HCV, maybe because inefficient RNA extraction as reported previously (Yang et al., 2011). CT values obtained for RNAseP from skin and nasopharyngeal swabs, respiratory specimens, and plasma extracted with Q were higher than with MP (Table 1B). The average CT difference calculated for each sample was positive for all sample types (Table 1B). We believe MP is a fast and effective method for NA extraction from nasopharyngeal and skin swabs, respiratory specimens and plasma. In contrast, the CT values for RNAseP were 5 cycles higher for blood samples extracted through MP compared with the salting-out method (Fig. 1B). Because hemoglobin present in blood inhibits PCR (Bessetti, 2007), we repurified MP-extracted DNAs with QIAGEN silica-column. However, CT values remained high (Fig. 1), maybe because extraction efficiency is low in blood specimens. We believe MP is not an appropriate method for NA extraction from blood for microbial diagnosis purposes. The choice of NA extraction method is a factor of major importance because it can impact diagnostic accuracy. In our location, the cost of MP per extraction is approximately 50% higher than Q. However, hands-on time is much higher for Q. We consider MP a highly convenient method for NA extraction of different kinds of pathogens from
NS
CT RNAse P
30
***
20
10
0 salting-out
MP
MP + Q
Fig. 1. CT values for human RNAseP amplification from blood samples extracted with the salting-out manual method, MP, and MP followed by purification with QIAGEN silica column (MP + Q). ***P b 0.0001, unpaired Student's t test between values of CTs from samples extracted with salting-out method and MP. NS, non-significant, paired Student's t test between values of CTs from samples extracted with MP and with MP + Q.
noncomplex specimens: its versatility, reduced risk of crosscontamination, and reduced hands-on time compensates its high cost. Acknowledgments This work was supported by research funds from the Department of Clinical Laboratories at Pontificia Universidad Católica de Chile. We acknowledge the personnel of Laboratorio de Microbiología, Pontificia Universidad Católica de Chile, for help with technical aspects of this work. The authors of this article declare they have no conflicts of interest. References Akutsu J, Tojo Y, Segawa O, Obata K, Okochi M, Tajima H, et al. Development of an integrated automation system with a magnetic bead-mediated nucleic acid purification device for genetic analysis and gene manipulation. Biotechnol Bioeng 2004;86: 667–71. Bessetti J. An introduction to PCR inhibitors. Profiles DNA 2007;10:9–10. Burd EM. Validation of laboratory-developed molecular assays for infectious diseases. Clin Microbiol Rev 2010;23:550–76. A. TK, Cowart KC, Winchell JM. Comparison-of-nucleic-acid-extraction-methods-for-thedetection-of-mycoplasma-pneumoniae. Diagn Microbiol Infect Dis 2009;65:435–8. Edvinsson B, Jalal S, Nord CE, Pedersen BS, Evengard B. DNA extraction and PCR assays for detection of Toxoplasma gondii. Apmis 2004;112:342–8. Goldschmidt P. S. Degorge, L. Merabet, and C. Chaumeil, 'Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious Agents'. PLoS One 2014;9, e94886. Ince J, McNally A. Development of rapid, automated diagnostics for infectious disease: advances and challenges. Expert Rev Med Devices 2009;6:641–51. Kosters K, Riffelmann M, Wirsing von Konig CH. Evaluation of a real-time PCR assay for detection of Bordetella pertussis and B. Parapertussis in clinical samples. J Med Microbiol 2001;50:436–40. L. PN, Elrod MG, Newton BR, Dauphin LA, Shi J, Chawalchitiporn S, et al. Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR. PLoS One 2013;8:e58032. Lahiri DK, Nurnberger Jr JI. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 1991;19:5444. Larsen HH, Masur H, Kovacs JA, Gill VJ, Silcott VA, Kogulan P, et al. Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia. J Clin Microbiol 2002;40:490–4. Luo W, Yang H, Rathbun K, Pau C-P, Chin-Yih O. Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay. J Clin Microbiol 2005;43:1851–7. Muldrew KL. Molecular diagnostics of infectious diseases. Curr Opin Pediatr 2009;21: 102–11. Yang JL, Wang MS, Cheng AC, Pan KC, Li CF, Deng SX. A simple and rapid method for extracting bacterial DNA from intestinal microflora for ERIC-PCR detection. World J Gastroenterol 2008;14:2872–6. Yang G, Erdman DE, Kodani M, Kools J, Bowen MD, Fields BS. Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens. J Virol Methods 2011;171:195–9. Yera H, Filisetti D, Bastien P, Ancelle T, Thulliez P, Delhaes L. Multicenter comparative evaluation of five commercial methods for toxoplasma DNA extraction from amniotic fluid. J Clin Microbiol 2009;47:3881–6.
Please cite this article as: Wozniak A, et al, Comparison of manual and automated nucleic acid extraction methods from clinical specimens for microbial diagnosis purposes, Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.07.008