Clinica Chimica Acta 445 (2015) 122–126
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A pressure-driven column-based technique for the efficient extraction of DNA from respiratory samples Takashi Hirama a,b,⁎, Hajime Mogi c, Hitoshi Egashira a, Eiji Yamamoto c, Shigenari Kukisaki c, Koichi Hagiwara a, Osamu Takei c a b c
Department of Respiratory Medicine, Saitama Medical University, 38 Morohongou, Moroyama, Saitama 350-0495, Japan Program in Cell Biology, Hospital for Sick Children, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Lifetech Co., Ltd, 4074 Miyadera, Iruma, Saitama, 358-0014, Japan
a r t i c l e
i n f o
Article history: Received 3 February 2015 Received in revised form 1 March 2015 Accepted 13 March 2015 Available online 27 March 2015 Keywords: Real-time PCR DNA extraction Pressure HIRA-TAN Column
a b s t r a c t Currently molecular techniques are a broadly accepted tool for diagnosis and are able to benefit patients in clinical practice. The polymerase chain reaction (PCR) has been especially incorporated into practical applications that are already in widespread use across the globe. With regard to the initial DNA extraction from clinically relevant samples, a number of commercially available kits are commonly used and are also designed to be easy to handle and less labor-intensive. In this study, the pressure system extracting DNA in column-based kit was developed, and its utility was compared with the centrifuge method using sputum from patients who were diagnosed with pneumonia. Also, due to the compact size and rapid processing time, the practical application of the pressure-based system incorporated into an automated pipetting machine was evaluated through clinical study. Our data suggests that DNA extraction by pressure was capable of serving as a substitute for the centrifuge method, and the compact and automatic nature of the pressure system device provided rapid and valuable information for clinical practice. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Currently molecular techniques have been involved in clinical practice and are already in widespread use throughout the globe. Notably, real-time PCR using respiratory specimens for identification of pathogens has been one of the significant clinical examinations [1,2]. Although DNA extraction from respiratory samples, sputum in particular, is by no means easy due to its viscosity. There are several kit options available for DNA extraction depending on the sample type [3–6]; however only column-based extraction has been used most for respiratory specimens [2,7–10]. Extracting DNA using spin columns requires a centrifuge as part of the process; however, the centrifuge is not a device that can be readily downsized. The vacuum procedure is also available but it requires an adequate means of waste disposal and special equipment. Hence, a conceivable alternative for the extraction of DNA from respiratory samples could be conducted by applying pressure to the column instead of centrifuging. Miniaturization and automation have been sought-after technologies in medical devices [11–13]. The more compact the instrument is the more acceptable and practical it becomes at the bedside. Likewise, ⁎ Corresponding author at: Department of Respiratory Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Saitama 350-0495, Japan. Tel./fax: +81 49 276 1319. E-mail address: [email protected] (T. Hirama).
http://dx.doi.org/10.1016/j.cca.2015.03.023 0009-8981/© 2015 Elsevier B.V. All rights reserved.
automatic equipment is able to provide the product with less intricacy, time and error, while complying with regulatory requirements. A newly developed DNA extraction method should therefore carry both of these benefits unlike existing methods. In this study, the pressure system extracting DNA in column-based kit was developed, and its utility was compared with the centrifuge method by using sputum from patients who were diagnosed with pneumonia. In addition, the system was incorporated into an automatic pipetting device, which was evaluated through clinical study. Our data suggests that DNA extraction by pressure was capable of serving as a substitute for the centrifuge, and the compact and automatic device installing the pressure system provided rapid and valuable information in clinical practice. 2. Materials and methods 2.1. Respiratory specimen (sputum) Sputum was collected from patients with pneumonia at the Saitama Medical University Hospital. The sample was mixed well by pipetting and divided into 2 parts; one was submitted for a regular bacteriological test (smear or culture) and the other for DNA extraction and real-time PCR analysis. To select purulent sputum, samples with an M2–P3 gross appearance were analyzed [1]. From each sample, an aliquot was stored at −80 °C for laboratory analysis.
T. Hirama et al. / Clinica Chimica Acta 445 (2015) 122–126
B
C 40
Pressure gauge
P 35 Cthuman
Hose pipe
Pressure syringe
Centrifuge Pressure
Spin column
30
Collection tube 25
60 DNA concentration (ng/µl)
A
123
Centrifuge Pressure
50 40 30 20 10
105 104 103 102 Cell number/tube
105 104 103 102 Cell number/tube
Fig. 1. Pilot experiment using the pressure-driven system. A. The scheme of manual pressurizer. The hand-made pressure was supplied from the pressure syringe to the column through a hosepipe and measured by pressure gauge. B. Relationship between cell number and Ct. Cultured A549 cells were suspended in DMEM and serially diluted. DNA was purified by the pressurizer and centrifuge methods, and a DNA sequence specific for human cell was amplified by real-time PCR. Experiments were performed at least three times. Error bars indicated mean ±SE. C. Relationship between cell number and DNA concentration. DNA was purified in the same manner as panel B. DNA concentration was measured based on DNA absorbance. Experiments were performed at least three times. Error bars indicated mean ± SE.
2.2. DNA preparation and quantification
2.5. Patients
The sample was diluted with an equal volume of phosphatebuffered saline (PBS) and homogenized by vortexing. Two hundred microliters of the homogenate was taken and mixed with 200 μl AL buffer (Qiagen, Tokyo, Japan) containing 20 μl proteinase K (Takara Bio Inc., Shiga, Japan), and the resultant mixture was incubated at 56 °C for 1 h. The protocols for pressure based or centrifuge based DNA extraction are described in Fig. 3. DNA was isolated into 100 or 500 μl of TE buffer with the QIAamp DNA Blood Mini Kit (Qiagen, Tokyo, Japan). Based on DNA absorbance, its concentration and purity were measured with a spectrophotometer GeneQuant Pro (GE healthcare, Tokyo, Japan). The ratio of nucleic acid to protein absorbance (260/280) was used as an indicator of the purity of DNA samples.
A diagnosis of pneumonia was clinically made when patients presented with both acutely produced/exacerbated illness, including cough, fever, sputum production, dyspnea, or chest discomfort/pain, and newly developed pulmonary infiltrates in a chest radiological examination without other alternative cause. Patients were eligible when they had pneumonia, were age 18 years or older and gave informed consent.
2.3. PCR reaction
3. Results
The final solution of the PCR reaction contained 12.5 μl of the TAKARA Premix Ex Taq (Takara Bio Inc., Shiga, Japan), 300 nM of each primer, 100 nM–300 nM of the fluorescence-labeled TaqMan probe, 1.0 μl of purified DNA and deionized distilled water up to 25.0 μl. The PCR was performed by starting at 95 °C for 30 s followed by 40 cycles at 95 °C for 8 s, 61 °C for 25 s, and 72 °C for 20 s using the SmartCycler II (Cepheid, Sunnyvale, CA). The sequences of primer and probe or its concentration were described in [2].
3.1. Applying pressure in place of a centrifuge to extract DNA
2.4. HIRA-TAN system We have adopted novel real-time PCR system named HIRA-TAN (Human cell-controlled Identification of the Respiratory Agent from “TAN” (which means sputum in Japanese)), which was able to discriminate therapeutic targets from commensal organisms (e.g. Streptococcus pneumoniae or Pseudomonas aeruginosa) and to detect foreign organisms (e.g. Mycoplasma pneumoniae or Mycobacterium tuberculosis) in the sputum. HIRA-TAN was capable of screening 23 target genes derived from respiratory pathogens in a lump and diagnosing the therapeutic target(s) among them. To determine the pathogenic role from commensal organisms detected by the real-time PCR, the HIRA-TAN system adopted ΔCtpathogen (difference between Cthuman and Ctpathogen), implying the pathogen to human cell number ratio. Through the previous projects, the ΔCtpathogen cutoff for each commensal organism was setup and successfully able to distinguish the therapeutic target from among detected commensal organisms. The technical details in the real-time PCR reaction and the HIRA-TAN system were discussed in more detail in [1,2].
2.6. Ethical consideration As to using patient's sample and the HIRA-TAN system for identification of respiratory pathogens, the research protocol was approved by the institutional review boards of the Saitama Medical University Hospital: IRB number 11-047 (February 8th, 2012).
In order to test our hypothesis, first we have designed a small pressurizer, which was manually able to apply pressure onto a column through a hosepipe with a 1.2 mm inner diameter (Fig. 1A). DNA extraction from cultured human cells using the pressurizer showed a comparable copy number and quantity of DNA as compared to the centrifuge (Fig. 1B, C). Secondly, using the identical sputum stored at −80 °C, we measured the extracted DNA concentration and purity, Cthuman [the human cell-specific gene with Ct (threshold cycle) value by real-time PCR] and identification concordance for the respiratory pathogen diagnosed by the HIRA-TAN system (see Materials and methods) between the hand-pressured and centrifuged method. These two approaches demonstrated similar results (Table 1), strongly indicating the potential of the pressure method as being one of the tools for DNA extraction.
Table 1 Comparison of manually hand-pressured and centrifuged method. Centrifuged extraction Hand-pressured extraction DNA concentration (ng/μl) DNA purity (260/280) Cthuman The number of pathogen detected by real-time PCR The number of pathogen diagnosed as therapeutic target
102.8 ± 93.8 1.67 ± 0.09 23.9 ± 2.2 12
91.8 ± 53.8 1.60 ± 0.11 23.0 ± 1.82 12 (1.0)
8
8 (1.0)
Data for DNA concentration and purity and Cthuman (Ct value for human cell specific gene) were mean ± SD. The concordance ratios for pathogen detection and diagnosis are shown in parentheses respectively. Data were collected from 11 stored sputum samples.
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Table 2 Flow volume for the pressure system. Flow volume (ml/min)
Cthuman
DNA concentration (ng/μl)
DNA purity (260 nm/280 nm)
5 30 150 300 Centrifuge
26.37 ± 0.44 25.99 ± 0.58 26.25 ± 0.13 25.98 ± 0.46 25.63 ± 0.05
37.43 ± 7.17 38.95 ± 5.98 33.98 ± 3.10 35.05 ± 6.49 69.65 ± 6.67
1.790 ± 0.017 1.795 ± 0.016 1.778 ± 0.028 1.760 ± 0.014 1.789 ± 0.094
Data for Cthuman and DNA concentration and purity were mean ± SD. Sputum was well homogenized and aliquoted into five tubes, and each flow volume was conducted. Data were collected from four stored sputum samples.
3.2. Development of the pressure system We next set the pressure force using the hosepipe. A series of flow volumes (3, 30, 150, and 300 ml/min) were examined and 30 ml/min was adopted as a standard pressing velocity because it demonstrated the most reproducible results (Table 2). The Cthuman represents the human cell number in the respiratory specimen and corresponds to the sputum's gross appearance [1], thus indicated as the most practical indicator for DNA extraction from sputum. 30 ml/min was continuously loaded until elution buffer containing DNA was passed through the column (around 5–10 s). Although we further studied the effect of varying pressing speed reaching at 30 ml/min, there was not much difference among them (Fig. 2). 3.3. Comparison of the performance between the pressure-based system and the centrifuge We subsequently compared and contrasted the performance of centrifuged extraction with the automated pressure extraction that was conducted by the pilot device to extract DNA (Supplemental Fig. 1A). Nineteen samples stored at −80 °C were analyzed according to the commercially available protocol or its modified protocol, respectively (Fig. 3). At STEP 12, extracting DNA with the pressure was implemented five times using as much volume of elution buffer (500 μl) as the centrifuge method. Alternatively, 5 μl DNA extract was applied to PCR reaction (STEP 14). The results from these two methods were quite similar, indicating that extracting DNA using pressure could be implemented as a replacement for the centrifuge method (Table 3). In addition, both showed 100% concordance for identification of pathogens and successfully discriminated the pathogenic role from identified pathogens by the HIRA-TAN system. 3.4. Automatic pipetting device
flow volume (ml/min)
3.4.1. Construction of the automatic pipetting device We therefore constructed the automatic pipetting device equipped with the pressure system, which was automatically able to carry out STEP 5 through STEP 15 in Fig. 3. After incubating the homogenate containing proteinase K and AL buffer at 56 °C (STEP 1–4), it was placed in the automatic pipetting device (Supplemental Fig. 1B, Movie 1) and
(A)
ethanol was then accordingly added to the sample. The device was miniaturized to a size of 826 mm high, 566 mm long and 600 mm wide, and had an operability to simultaneously extract DNA from four samples in a single assay and split it into separate PCR reaction tubes. In order to reduce error, the automatic pipette machine eluted DNA by 5 times buffer and then 5 μl of the DNA sample was added into each reaction tube. Consumables, such as sputum homogenates, PCR reaction solutions containing primers and a probe, 20 μl and 1000 μl pipette tips and 25 μl SmartCycler reaction tubes were situated for ease of access in order to dispense samples (Supplemental Fig. 1C). 20 μl and 1000 μl pipettes, the agitator and the pressure system were placed to function efficiently (Supplemental Fig. 1B, C). 3.4.2. Clinical study We compared the ability of the pressure-based DNA extraction equipped in the automatic pipetting device with the centrifuge base using clinical samples. Fifty patients who were diagnosed with pneumonia at Saitama Medical University Hospital were serially enrolled for the analysis and the results of the extract and HIRA-TAN system were summarized in Table 4. The copy number of human cell contained in the sputum, together with the purity of extracted DNA, was comparable between these assays. Also, while the bacterial test cultured the causative pathogens, both methods detected the same pathogens with similar Ctpathogen. In addition, the HIRA-TAN system could diagnose the identical pathogen with comparable ΔCtpathogen in clinical study, indicating that the pressure-based DNA extraction showed functional equality to the centrifuge-based system and could be a feasible approach for realtime PCR-based diagnosis. Moreover the pressure system was small enough so that we could install it into the automatic pipetting device, which also enabled us to minimize the whole device size. 4. Discussion DNA extraction from clinical samples using the column-based kit has been highly popularized for molecular diagnosis. This work thus set out to investigate whether applying pressure could be a method to replace the centrifuge in the DNA extraction process and to evaluate its utility. In addition, due to the compact size and rapid processing time, the practical application of the pressure-based system incorporated into an automated pipetting machine was evaluated through clinical study. Respiratory specimens, such as sputum, can be the most cumbersome to extract nucleic acid due to its viscous property relative to other clinical specimens. Since the pressure-based system worked out on sputa, it would be able to be implemented in other clinical samples, such as blood, cerebrospinal fluid, urine or feces, indicating a wider application in a variety of clinical settings. More practical medical devices require the improvement of performance through the occupation of less space, more rapid processing times and, preferably, automation by highly modified versions of existing devices. In terms of extracting nucleic acid, the centrifuge device is necessary on its process since the column-based kits have been widely accepted as a standard method. However, such a device requires valuable space, which has prevented miniaturization of whole instrument size. The pressuring system designed in this study was palm-size.
(B)
(C)
30
time
time
time
Fig. 2. Scheme of pressing speed examined. Velocity pressing (A) rapidly, (B) intermediately and (C) gradually to achieve at 30 ml/min were administrated.
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125
Protocol for centrifuge-based extraction
Protocol for pressure-based extraction
Place 1.5 ml microcentrifuge tube
Place 1.5 ml microcentrifuge tube
1. 2. 3. 4. 5. 6.
1. 2. 3. 4. 5. 6.
Pipet 20 µl proteinase K into the tube Add 200 µl homogenate sample Add 200 µl AL buffer to the homogenate Incubate the tube at 56°C for 60 min Add 200 µl 96% ethanol to the homogenate Apply the mixture to the QIAamp Mini spin column
Pipet 20 µl proteinase K into the tube Add 200 µl homogenate sample Add 200 µl AL buffer to the homogenate Incubate the tube at 56°C for 60 min Add 200 µl 96% ethanol to the homogenate Apply the mixture to the QIAamp Mini spin column
Place the column in 2.0 ml microcentrifuge tube 7. Centrifuge at 8000 rpm for 1 min 8. Add 500 µl AW1 buffer 9. Centrifuge at 8000 rpm for 1 min 10. Add 500 µl AW2 buffer 11. Centrifuge at 14,000 rpm for 3 min
Place the column in 1.5 ml microcentrifuge tube 7. Pressure at 30 ml/min 8. Add 500 µl AW1 buffer 9. Pressure at 30 ml/min 10. Add 500 µl AW2 buffer 11. Pressure at 30 ml/min
Place the column in 2.0 ml microcentrifuge tube
Place the column in 1.5 ml microcentrifuge tube
12. 13. 14. 15. 16.
12. 13. 14. 15. 16.
Add 100 µl AE buffer Centrifuge at 8000 rpm for 1 min Apply 1 µl extracted DNA to the PCR tubes Add 24 µl PCR reaction solution to DNA Implement real-time PCR
Add 500 µl AE buffer Pressure at 30 ml/min Apply 5 µl extracted DNA to the PCR tubes Add 20 µl PCR reaction solution to DNA Implement real-time PCR
Fig. 3. Protocols of DNA extraction for the centrifuge and pressure. Protocol for centrifuge-based extraction was summarized according to the commercialized product. Protocol for pressure-based extraction was modified on the basis of automatic pipetting device. Dashed rectangle indicated the automated process.
Accordingly, applying this system enabled the instrument size minimum and would be easier to perform than that of the centrifuge device or vacuum approach as described in the manufacturer's protocols. In addition, this system was able to not only function at a rapid pace but also to avoid contamination with other templates and PCR products that might be present in the laboratory environment and to limit exposure of examiners to aerosol transmission and/or infectious agents. Furthermore, the precision automation gave processing time within 3 h to report the therapeutic targets in pneumonia by the HIRA-TAN system. On the other hand, DNA extracting process shown in Fig. 3 was capable of being incorporated into the automatic pipetting device, which was designed implementing STEP 5–15. Given the requirement of the biosafety cabinet in STEP 1–4, automation from ethanol precipitation (STEP 5) should have flexible options for a variety of samples.
The most prominent feature of sputum is its viscosity and it can interfere with extracting DNA and conducting molecular diagnosis smoothly. The Ct value is defined as the number of cycles at which significant fluorescent signal is initially observed. Therefore, Cthuman in sputum represents the cell number of polymorphonuclear leukocytes or respiratory epithelium, and corresponds to the degree of sputum purulence and total DNA concentration [1,14]. Although some highly viscous sputum samples lack correlation between DNA concentration and Cthuman in the current study, the Ct values in both the centrifuge and the pressure system illustrated a similar finding. Likewise, with samples treated in the both methods, the HIRA-TAN system was able to demonstrate 100% concordance to identify the causative pathogen from the detected organisms in clinical study. Meanwhile, highly viscous sputum caused column clogging, suggesting the limitation of
Table 3 HIRA-TAN results from centrifuged and pressure-based system. Centrifuged-based system
Pressure-based system
Sample
Purulence
Identified pathogen
Cthuman
Ctpathogen
ΔCt
Pathogenic role
Identified pathogen
Cthuman
Ctpathogen
ΔCt
Pathogenic role
001 002 003 004 005 006 007
P2 P2 P3 P3 P3 M2 P1
24.35 22.27 23.17 22.2 26.97 30.24 28.2
P3 P3 P3 P3 P3 P3 P2 P1 P3 P3 P3 M2
20.11 17.12 24.33 25.24 29.21 38.56 37.22 30.03 35.26 18.46 22.19 27.28 20.31 20.78 25.56 17.18 23.03 29.05 22.58 24.31
4.24 5.15 −1.16 −3.04 −2.24 −8.32 −9.02 −1.83 −13.11 5.02 2.03 −4.03 3.94 1.85 −2.09 8.11 0.85 −4.67 3.11 0.6
Causative Causative Colonizing Causative Causative Colonizing Colonizing Colonizing Colonizing Causative Causative Causative Causative Causative Causative Causative Causative Colonizing Causative Causative
H inf S pne M cat S pne S pne S pne S pne M cat S pne S pne S pne S pne K pne S pne S pne P aer K pne P aer P aer P aer
22.42 22.4 21.68 21.26 22.61 29.78 27.58
008 009 010 011 012 013 014 015 016 017 018 019
H inf S pne M cat S pne S pne S pne S pne M cat S pne S pne S pne S pne K pne S pne S pne P aer K pne P aer P aer P aer
18.03 17.39 22.93 25.08 26.16 34.44 36.55 30.42 34.16 18.5 22.09 27.72 22.84 20.37 25.04 16.76 22.75 28.41 22.23 24.04
4.39 5.01 −1.25 −3.82 −3.55 −4.66 −8.97 −2.84 −9.25 5.17 2.07 −3.87 3.39 1.99 −2.16 8.45 1.75 −4.61 3.02 0.6
Causative Causative Colonizing Causative Causative Colonizing Colonizing Colonizing Colonizing Causative Causative Causative Causative Causative Causative Causative Causative Colonizing Causative Causative
22.15 23.48 24.22 23.25 24.25 22.63 23.47 25.29 23.88 24.38 25.69 24.91
24.91 23.67 24.16 23.85 26.23 22.36 22.88 25.21 24.5 23.8 25.25 24.64
Abbreviations of pathogens were S pne, Streptococcus pneumoniae; H inf, Haemophilus influenzae; M cat, Moraxella catarrhalis; P aer, Pseudomonas aeruginosa and K pne, Klebsiella pneumonia. ΔCt (Cthuman − Ctpathogen) represented the cut-off value to discriminate the pathogenic role from identified pathogens in the HIRA-TAN system, which was − 6 for S pne; −1 for H inf; −4 for P aer; −6 for K pne and 0 for M cat [RespMed2014]. Data were collected from 19 stored sputum samples.
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Table 4 Summary of the clinical study with pressure-based system equipped in the automated pipetting device. DNA extraction method
Centrifuge-based system
Pressure-based system
Overview of the devices Flow volume Sample volume Elution buffer PCR reaction solution Template DNA
– 200 μl 100 μl 24 μl 1 μl
30 ml/min 200 μl 500 μl 20 μl 5 μl
Results of clinical samples Cthuman DNA purity (260 nm/280 nm) Therapeutic target in commensal organisms Therapeutic target in foreign organisms Average time taken to perform STEP 5–15
24.05 ± 2.33 1.76 ± 0.11 29 2 1–2 h
24.11 ± 2.02 1.73 ± 0.14 29 (1.0) 2 (1.0) 0.5–1 h
Sputum was collected from patients who were diagnosed with pneumonia and analyzed as soon as expectorated. Data for Cthuman and DNA purity were mean ± SD. The concordance ratios for diagnosis of therapeutic target in commensal organisms and foreign organisms in HIRA-TAN system were shown in parentheses respectively (see Materials and methods). Data were collected from 50 sputum samples through clinical study.
filtration of the respiratory specimen at the spin column. As sputum viscosity was independent from gross appearance or DNA concentration [14], the sputum should be considered as a totally different specimen from other clinical samples. We coped with column clogging due to high viscosity by applying pressure twice whereby DNA was extractable. Despite the troubling aspect of sputum's viscosity, the pressurebased system could extract DNA and show proper Cthuman in all samples. Although, what caused small Cthuman from high viscosity samples needs to be further studied, the pressure system could be applied to the molecular diagnosis by using respiratory specimens. In conclusion, the pressure system was able to take the place of the centrifuge in a respiratory specimen and rapidly provide information to the bedside. Moreover, the pressure system yielded the space and time benefits of processing clinical samples and would promote the treatment of pneumonia by combining automatic pipetting device. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2015.03.023. Disclosure The authors have declared that no competing interests exist. This work was supported by the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (AS2321117F) from the Japan Science and Technology Agency and Grant-in-Aid for Young Scientists (B) (No. 23790918) from the Japan Society for the Promotion of Science. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. In addition, the manuscript has been reviewed by all of the authors and is not being
submitted to any other journals. All of the authors have declared that no competing interests exist. Acknowledgments For their collaboration and the enrollment of their patients, we are indebted to Makoto Nagata, MD, PhD and Minoru Kanazawa, MD, PhD, the Department of Respiratory Medicine, Saitama Medical University, Saitama and Etsuko Kishi, the Central Laboratory, Saitama Medical University, Saitama We would like to thank my colleague Johnathan Canton, PhD, Program in Cell Biology, Hospital for Sick Children, Toronto, Canada, for proofreading of manuscript draft. References [1] Hirama T, Yamaguchi T, Miyazawa H, et al. Prediction of the pathogens that are the cause of pneumonia by the battlefield hypothesis. PLoS One 2011;6:e24474. http://dx.doi.org/10.1371/journal.pone.0024474. [2] Hirama T, Minezaki S, Yamaguchi T, et al. HIRA-TAN: a real-time PCR-based system for the rapid identification of causative agents in pneumonia. Respir Med 2014;108: 395. http://dx.doi.org/10.1016/j.rmed.2013.11.018. [3] Wilson D, Yen-Lieberman B, Reischl U, Warshawsky I, Procop GW. Comparison of five methods for extraction of Legionella pneumophila from respiratory specimens. J Clin Microbiol 2004;42:5913–6. http://dx.doi.org/10.1128/JCM. 42.12.5913-5916. 2004. [4] Aldous WK, Pounder JI, Cloud JL, Woods GL. Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR. J Clin Microbiol 2005;43:2471–3. http://dx.doi.org/10.1128/JCM. 43.5. 2471-2473.2005. [5] Queipo-Ortuño MI, Tena F, Colmenero JD, Morata P. 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; 27:109–14. http://dx.doi.org/10.1007/s10096-007-0409-y. [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. http://dx.doi.org/10.1016/j.jviromet. 2010.10.024. [7] Deng J, Ma Z, Huang W, et al. Respiratory virus multiplex RT-PCR assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections. Virol Sin 2013;28:97–102. http://dx.doi.org/10.1007/s12250-013-3312-y. [8] Li J, Mao N-Y, Zhang C, et al. The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes. BMC Infect Dis 2012;12:189. http://dx.doi.org/10.1186/14712334-12-189. [9] Chotirmall SH, McElvaney NG. Fungi in the cystic fibrosis lung: bystanders or pathogens? Int J Biochem Cell Biol 2014;52:161–73. http://dx.doi.org/10.1016/j. biocel.2014.03.001. [10] Bouchara J-P, Hsieh HY, Croquefer S, et al. Development of an oligonucleotide array for direct detection of fungi in sputum samples from patients with cystic fibrosis. J Clin Microbiol 2009;47:142–52. http://dx.doi.org/10.1128/JCM. 01668-08. [11] Zhang Y, Zhu Y, Yao B, Fang Q. Nanolitre droplet array for real time reverse transcription polymerase chain reaction. Lab Chip 2011;11:1545–9. http://dx.doi.org/10. 1039/c0lc00502a. [12] Hwang K-Y, Kwon SH, Jung S-O, et al. Miniaturized bead-beating device to automate full DNA sample preparation processes for gram-positive bacteria. Lab Chip 2011;11: 3649–55. http://dx.doi.org/10.1039/c1lc20692c. [13] Lee AV, Atkinson C, Manuel RJ, Clark DA. Comparative evaluation of the QIAGEN QIAsymphony® SP system and bioMérieux NucliSens easyMAG automated extraction platforms in a clinical virology laboratory. J Clin Virol 2011;52:339–43. http://dx.doi.org/10.1016/j.jcv.2011.08.016. [14] Picot R, Das I, Reid L. Pus, deoxyribonucleic acid, and sputum viscosity. Thorax 1978; 33:235–42. http://dx.doi.org/10.1136/thx.33.2.235.
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A pressure-driven column-based technique for the efficient extraction of DNA from respiratory samples Takashi Hirama a,b,⁎, Hajime Mogi c, Hitoshi Egashira a, Eiji Yamamoto c, Shigenari Kukisaki c, Koichi Hagiwara a, Osamu Takei c a b c
Department of Respiratory Medicine, Saitama Medical University, 38 Morohongou, Moroyama, Saitama 350-0495, Japan Program in Cell Biology, Hospital for Sick Children, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Lifetech Co., Ltd, 4074 Miyadera, Iruma, Saitama, 358-0014, Japan
a r t i c l e
i n f o
Article history: Received 3 February 2015 Received in revised form 1 March 2015 Accepted 13 March 2015 Available online 27 March 2015 Keywords: Real-time PCR DNA extraction Pressure HIRA-TAN Column
a b s t r a c t Currently molecular techniques are a broadly accepted tool for diagnosis and are able to benefit patients in clinical practice. The polymerase chain reaction (PCR) has been especially incorporated into practical applications that are already in widespread use across the globe. With regard to the initial DNA extraction from clinically relevant samples, a number of commercially available kits are commonly used and are also designed to be easy to handle and less labor-intensive. In this study, the pressure system extracting DNA in column-based kit was developed, and its utility was compared with the centrifuge method using sputum from patients who were diagnosed with pneumonia. Also, due to the compact size and rapid processing time, the practical application of the pressure-based system incorporated into an automated pipetting machine was evaluated through clinical study. Our data suggests that DNA extraction by pressure was capable of serving as a substitute for the centrifuge method, and the compact and automatic nature of the pressure system device provided rapid and valuable information for clinical practice. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Currently molecular techniques have been involved in clinical practice and are already in widespread use throughout the globe. Notably, real-time PCR using respiratory specimens for identification of pathogens has been one of the significant clinical examinations [1,2]. Although DNA extraction from respiratory samples, sputum in particular, is by no means easy due to its viscosity. There are several kit options available for DNA extraction depending on the sample type [3–6]; however only column-based extraction has been used most for respiratory specimens [2,7–10]. Extracting DNA using spin columns requires a centrifuge as part of the process; however, the centrifuge is not a device that can be readily downsized. The vacuum procedure is also available but it requires an adequate means of waste disposal and special equipment. Hence, a conceivable alternative for the extraction of DNA from respiratory samples could be conducted by applying pressure to the column instead of centrifuging. Miniaturization and automation have been sought-after technologies in medical devices [11–13]. The more compact the instrument is the more acceptable and practical it becomes at the bedside. Likewise, ⁎ Corresponding author at: Department of Respiratory Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Saitama 350-0495, Japan. Tel./fax: +81 49 276 1319. E-mail address: [email protected] (T. Hirama).
http://dx.doi.org/10.1016/j.cca.2015.03.023 0009-8981/© 2015 Elsevier B.V. All rights reserved.
automatic equipment is able to provide the product with less intricacy, time and error, while complying with regulatory requirements. A newly developed DNA extraction method should therefore carry both of these benefits unlike existing methods. In this study, the pressure system extracting DNA in column-based kit was developed, and its utility was compared with the centrifuge method by using sputum from patients who were diagnosed with pneumonia. In addition, the system was incorporated into an automatic pipetting device, which was evaluated through clinical study. Our data suggests that DNA extraction by pressure was capable of serving as a substitute for the centrifuge, and the compact and automatic device installing the pressure system provided rapid and valuable information in clinical practice. 2. Materials and methods 2.1. Respiratory specimen (sputum) Sputum was collected from patients with pneumonia at the Saitama Medical University Hospital. The sample was mixed well by pipetting and divided into 2 parts; one was submitted for a regular bacteriological test (smear or culture) and the other for DNA extraction and real-time PCR analysis. To select purulent sputum, samples with an M2–P3 gross appearance were analyzed [1]. From each sample, an aliquot was stored at −80 °C for laboratory analysis.
T. Hirama et al. / Clinica Chimica Acta 445 (2015) 122–126
B
C 40
Pressure gauge
P 35 Cthuman
Hose pipe
Pressure syringe
Centrifuge Pressure
Spin column
30
Collection tube 25
60 DNA concentration (ng/µl)
A
123
Centrifuge Pressure
50 40 30 20 10
105 104 103 102 Cell number/tube
105 104 103 102 Cell number/tube
Fig. 1. Pilot experiment using the pressure-driven system. A. The scheme of manual pressurizer. The hand-made pressure was supplied from the pressure syringe to the column through a hosepipe and measured by pressure gauge. B. Relationship between cell number and Ct. Cultured A549 cells were suspended in DMEM and serially diluted. DNA was purified by the pressurizer and centrifuge methods, and a DNA sequence specific for human cell was amplified by real-time PCR. Experiments were performed at least three times. Error bars indicated mean ±SE. C. Relationship between cell number and DNA concentration. DNA was purified in the same manner as panel B. DNA concentration was measured based on DNA absorbance. Experiments were performed at least three times. Error bars indicated mean ± SE.
2.2. DNA preparation and quantification
2.5. Patients
The sample was diluted with an equal volume of phosphatebuffered saline (PBS) and homogenized by vortexing. Two hundred microliters of the homogenate was taken and mixed with 200 μl AL buffer (Qiagen, Tokyo, Japan) containing 20 μl proteinase K (Takara Bio Inc., Shiga, Japan), and the resultant mixture was incubated at 56 °C for 1 h. The protocols for pressure based or centrifuge based DNA extraction are described in Fig. 3. DNA was isolated into 100 or 500 μl of TE buffer with the QIAamp DNA Blood Mini Kit (Qiagen, Tokyo, Japan). Based on DNA absorbance, its concentration and purity were measured with a spectrophotometer GeneQuant Pro (GE healthcare, Tokyo, Japan). The ratio of nucleic acid to protein absorbance (260/280) was used as an indicator of the purity of DNA samples.
A diagnosis of pneumonia was clinically made when patients presented with both acutely produced/exacerbated illness, including cough, fever, sputum production, dyspnea, or chest discomfort/pain, and newly developed pulmonary infiltrates in a chest radiological examination without other alternative cause. Patients were eligible when they had pneumonia, were age 18 years or older and gave informed consent.
2.3. PCR reaction
3. Results
The final solution of the PCR reaction contained 12.5 μl of the TAKARA Premix Ex Taq (Takara Bio Inc., Shiga, Japan), 300 nM of each primer, 100 nM–300 nM of the fluorescence-labeled TaqMan probe, 1.0 μl of purified DNA and deionized distilled water up to 25.0 μl. The PCR was performed by starting at 95 °C for 30 s followed by 40 cycles at 95 °C for 8 s, 61 °C for 25 s, and 72 °C for 20 s using the SmartCycler II (Cepheid, Sunnyvale, CA). The sequences of primer and probe or its concentration were described in [2].
3.1. Applying pressure in place of a centrifuge to extract DNA
2.4. HIRA-TAN system We have adopted novel real-time PCR system named HIRA-TAN (Human cell-controlled Identification of the Respiratory Agent from “TAN” (which means sputum in Japanese)), which was able to discriminate therapeutic targets from commensal organisms (e.g. Streptococcus pneumoniae or Pseudomonas aeruginosa) and to detect foreign organisms (e.g. Mycoplasma pneumoniae or Mycobacterium tuberculosis) in the sputum. HIRA-TAN was capable of screening 23 target genes derived from respiratory pathogens in a lump and diagnosing the therapeutic target(s) among them. To determine the pathogenic role from commensal organisms detected by the real-time PCR, the HIRA-TAN system adopted ΔCtpathogen (difference between Cthuman and Ctpathogen), implying the pathogen to human cell number ratio. Through the previous projects, the ΔCtpathogen cutoff for each commensal organism was setup and successfully able to distinguish the therapeutic target from among detected commensal organisms. The technical details in the real-time PCR reaction and the HIRA-TAN system were discussed in more detail in [1,2].
2.6. Ethical consideration As to using patient's sample and the HIRA-TAN system for identification of respiratory pathogens, the research protocol was approved by the institutional review boards of the Saitama Medical University Hospital: IRB number 11-047 (February 8th, 2012).
In order to test our hypothesis, first we have designed a small pressurizer, which was manually able to apply pressure onto a column through a hosepipe with a 1.2 mm inner diameter (Fig. 1A). DNA extraction from cultured human cells using the pressurizer showed a comparable copy number and quantity of DNA as compared to the centrifuge (Fig. 1B, C). Secondly, using the identical sputum stored at −80 °C, we measured the extracted DNA concentration and purity, Cthuman [the human cell-specific gene with Ct (threshold cycle) value by real-time PCR] and identification concordance for the respiratory pathogen diagnosed by the HIRA-TAN system (see Materials and methods) between the hand-pressured and centrifuged method. These two approaches demonstrated similar results (Table 1), strongly indicating the potential of the pressure method as being one of the tools for DNA extraction.
Table 1 Comparison of manually hand-pressured and centrifuged method. Centrifuged extraction Hand-pressured extraction DNA concentration (ng/μl) DNA purity (260/280) Cthuman The number of pathogen detected by real-time PCR The number of pathogen diagnosed as therapeutic target
102.8 ± 93.8 1.67 ± 0.09 23.9 ± 2.2 12
91.8 ± 53.8 1.60 ± 0.11 23.0 ± 1.82 12 (1.0)
8
8 (1.0)
Data for DNA concentration and purity and Cthuman (Ct value for human cell specific gene) were mean ± SD. The concordance ratios for pathogen detection and diagnosis are shown in parentheses respectively. Data were collected from 11 stored sputum samples.
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Table 2 Flow volume for the pressure system. Flow volume (ml/min)
Cthuman
DNA concentration (ng/μl)
DNA purity (260 nm/280 nm)
5 30 150 300 Centrifuge
26.37 ± 0.44 25.99 ± 0.58 26.25 ± 0.13 25.98 ± 0.46 25.63 ± 0.05
37.43 ± 7.17 38.95 ± 5.98 33.98 ± 3.10 35.05 ± 6.49 69.65 ± 6.67
1.790 ± 0.017 1.795 ± 0.016 1.778 ± 0.028 1.760 ± 0.014 1.789 ± 0.094
Data for Cthuman and DNA concentration and purity were mean ± SD. Sputum was well homogenized and aliquoted into five tubes, and each flow volume was conducted. Data were collected from four stored sputum samples.
3.2. Development of the pressure system We next set the pressure force using the hosepipe. A series of flow volumes (3, 30, 150, and 300 ml/min) were examined and 30 ml/min was adopted as a standard pressing velocity because it demonstrated the most reproducible results (Table 2). The Cthuman represents the human cell number in the respiratory specimen and corresponds to the sputum's gross appearance [1], thus indicated as the most practical indicator for DNA extraction from sputum. 30 ml/min was continuously loaded until elution buffer containing DNA was passed through the column (around 5–10 s). Although we further studied the effect of varying pressing speed reaching at 30 ml/min, there was not much difference among them (Fig. 2). 3.3. Comparison of the performance between the pressure-based system and the centrifuge We subsequently compared and contrasted the performance of centrifuged extraction with the automated pressure extraction that was conducted by the pilot device to extract DNA (Supplemental Fig. 1A). Nineteen samples stored at −80 °C were analyzed according to the commercially available protocol or its modified protocol, respectively (Fig. 3). At STEP 12, extracting DNA with the pressure was implemented five times using as much volume of elution buffer (500 μl) as the centrifuge method. Alternatively, 5 μl DNA extract was applied to PCR reaction (STEP 14). The results from these two methods were quite similar, indicating that extracting DNA using pressure could be implemented as a replacement for the centrifuge method (Table 3). In addition, both showed 100% concordance for identification of pathogens and successfully discriminated the pathogenic role from identified pathogens by the HIRA-TAN system. 3.4. Automatic pipetting device
flow volume (ml/min)
3.4.1. Construction of the automatic pipetting device We therefore constructed the automatic pipetting device equipped with the pressure system, which was automatically able to carry out STEP 5 through STEP 15 in Fig. 3. After incubating the homogenate containing proteinase K and AL buffer at 56 °C (STEP 1–4), it was placed in the automatic pipetting device (Supplemental Fig. 1B, Movie 1) and
(A)
ethanol was then accordingly added to the sample. The device was miniaturized to a size of 826 mm high, 566 mm long and 600 mm wide, and had an operability to simultaneously extract DNA from four samples in a single assay and split it into separate PCR reaction tubes. In order to reduce error, the automatic pipette machine eluted DNA by 5 times buffer and then 5 μl of the DNA sample was added into each reaction tube. Consumables, such as sputum homogenates, PCR reaction solutions containing primers and a probe, 20 μl and 1000 μl pipette tips and 25 μl SmartCycler reaction tubes were situated for ease of access in order to dispense samples (Supplemental Fig. 1C). 20 μl and 1000 μl pipettes, the agitator and the pressure system were placed to function efficiently (Supplemental Fig. 1B, C). 3.4.2. Clinical study We compared the ability of the pressure-based DNA extraction equipped in the automatic pipetting device with the centrifuge base using clinical samples. Fifty patients who were diagnosed with pneumonia at Saitama Medical University Hospital were serially enrolled for the analysis and the results of the extract and HIRA-TAN system were summarized in Table 4. The copy number of human cell contained in the sputum, together with the purity of extracted DNA, was comparable between these assays. Also, while the bacterial test cultured the causative pathogens, both methods detected the same pathogens with similar Ctpathogen. In addition, the HIRA-TAN system could diagnose the identical pathogen with comparable ΔCtpathogen in clinical study, indicating that the pressure-based DNA extraction showed functional equality to the centrifuge-based system and could be a feasible approach for realtime PCR-based diagnosis. Moreover the pressure system was small enough so that we could install it into the automatic pipetting device, which also enabled us to minimize the whole device size. 4. Discussion DNA extraction from clinical samples using the column-based kit has been highly popularized for molecular diagnosis. This work thus set out to investigate whether applying pressure could be a method to replace the centrifuge in the DNA extraction process and to evaluate its utility. In addition, due to the compact size and rapid processing time, the practical application of the pressure-based system incorporated into an automated pipetting machine was evaluated through clinical study. Respiratory specimens, such as sputum, can be the most cumbersome to extract nucleic acid due to its viscous property relative to other clinical specimens. Since the pressure-based system worked out on sputa, it would be able to be implemented in other clinical samples, such as blood, cerebrospinal fluid, urine or feces, indicating a wider application in a variety of clinical settings. More practical medical devices require the improvement of performance through the occupation of less space, more rapid processing times and, preferably, automation by highly modified versions of existing devices. In terms of extracting nucleic acid, the centrifuge device is necessary on its process since the column-based kits have been widely accepted as a standard method. However, such a device requires valuable space, which has prevented miniaturization of whole instrument size. The pressuring system designed in this study was palm-size.
(B)
(C)
30
time
time
time
Fig. 2. Scheme of pressing speed examined. Velocity pressing (A) rapidly, (B) intermediately and (C) gradually to achieve at 30 ml/min were administrated.
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Protocol for centrifuge-based extraction
Protocol for pressure-based extraction
Place 1.5 ml microcentrifuge tube
Place 1.5 ml microcentrifuge tube
1. 2. 3. 4. 5. 6.
1. 2. 3. 4. 5. 6.
Pipet 20 µl proteinase K into the tube Add 200 µl homogenate sample Add 200 µl AL buffer to the homogenate Incubate the tube at 56°C for 60 min Add 200 µl 96% ethanol to the homogenate Apply the mixture to the QIAamp Mini spin column
Pipet 20 µl proteinase K into the tube Add 200 µl homogenate sample Add 200 µl AL buffer to the homogenate Incubate the tube at 56°C for 60 min Add 200 µl 96% ethanol to the homogenate Apply the mixture to the QIAamp Mini spin column
Place the column in 2.0 ml microcentrifuge tube 7. Centrifuge at 8000 rpm for 1 min 8. Add 500 µl AW1 buffer 9. Centrifuge at 8000 rpm for 1 min 10. Add 500 µl AW2 buffer 11. Centrifuge at 14,000 rpm for 3 min
Place the column in 1.5 ml microcentrifuge tube 7. Pressure at 30 ml/min 8. Add 500 µl AW1 buffer 9. Pressure at 30 ml/min 10. Add 500 µl AW2 buffer 11. Pressure at 30 ml/min
Place the column in 2.0 ml microcentrifuge tube
Place the column in 1.5 ml microcentrifuge tube
12. 13. 14. 15. 16.
12. 13. 14. 15. 16.
Add 100 µl AE buffer Centrifuge at 8000 rpm for 1 min Apply 1 µl extracted DNA to the PCR tubes Add 24 µl PCR reaction solution to DNA Implement real-time PCR
Add 500 µl AE buffer Pressure at 30 ml/min Apply 5 µl extracted DNA to the PCR tubes Add 20 µl PCR reaction solution to DNA Implement real-time PCR
Fig. 3. Protocols of DNA extraction for the centrifuge and pressure. Protocol for centrifuge-based extraction was summarized according to the commercialized product. Protocol for pressure-based extraction was modified on the basis of automatic pipetting device. Dashed rectangle indicated the automated process.
Accordingly, applying this system enabled the instrument size minimum and would be easier to perform than that of the centrifuge device or vacuum approach as described in the manufacturer's protocols. In addition, this system was able to not only function at a rapid pace but also to avoid contamination with other templates and PCR products that might be present in the laboratory environment and to limit exposure of examiners to aerosol transmission and/or infectious agents. Furthermore, the precision automation gave processing time within 3 h to report the therapeutic targets in pneumonia by the HIRA-TAN system. On the other hand, DNA extracting process shown in Fig. 3 was capable of being incorporated into the automatic pipetting device, which was designed implementing STEP 5–15. Given the requirement of the biosafety cabinet in STEP 1–4, automation from ethanol precipitation (STEP 5) should have flexible options for a variety of samples.
The most prominent feature of sputum is its viscosity and it can interfere with extracting DNA and conducting molecular diagnosis smoothly. The Ct value is defined as the number of cycles at which significant fluorescent signal is initially observed. Therefore, Cthuman in sputum represents the cell number of polymorphonuclear leukocytes or respiratory epithelium, and corresponds to the degree of sputum purulence and total DNA concentration [1,14]. Although some highly viscous sputum samples lack correlation between DNA concentration and Cthuman in the current study, the Ct values in both the centrifuge and the pressure system illustrated a similar finding. Likewise, with samples treated in the both methods, the HIRA-TAN system was able to demonstrate 100% concordance to identify the causative pathogen from the detected organisms in clinical study. Meanwhile, highly viscous sputum caused column clogging, suggesting the limitation of
Table 3 HIRA-TAN results from centrifuged and pressure-based system. Centrifuged-based system
Pressure-based system
Sample
Purulence
Identified pathogen
Cthuman
Ctpathogen
ΔCt
Pathogenic role
Identified pathogen
Cthuman
Ctpathogen
ΔCt
Pathogenic role
001 002 003 004 005 006 007
P2 P2 P3 P3 P3 M2 P1
24.35 22.27 23.17 22.2 26.97 30.24 28.2
P3 P3 P3 P3 P3 P3 P2 P1 P3 P3 P3 M2
20.11 17.12 24.33 25.24 29.21 38.56 37.22 30.03 35.26 18.46 22.19 27.28 20.31 20.78 25.56 17.18 23.03 29.05 22.58 24.31
4.24 5.15 −1.16 −3.04 −2.24 −8.32 −9.02 −1.83 −13.11 5.02 2.03 −4.03 3.94 1.85 −2.09 8.11 0.85 −4.67 3.11 0.6
Causative Causative Colonizing Causative Causative Colonizing Colonizing Colonizing Colonizing Causative Causative Causative Causative Causative Causative Causative Causative Colonizing Causative Causative
H inf S pne M cat S pne S pne S pne S pne M cat S pne S pne S pne S pne K pne S pne S pne P aer K pne P aer P aer P aer
22.42 22.4 21.68 21.26 22.61 29.78 27.58
008 009 010 011 012 013 014 015 016 017 018 019
H inf S pne M cat S pne S pne S pne S pne M cat S pne S pne S pne S pne K pne S pne S pne P aer K pne P aer P aer P aer
18.03 17.39 22.93 25.08 26.16 34.44 36.55 30.42 34.16 18.5 22.09 27.72 22.84 20.37 25.04 16.76 22.75 28.41 22.23 24.04
4.39 5.01 −1.25 −3.82 −3.55 −4.66 −8.97 −2.84 −9.25 5.17 2.07 −3.87 3.39 1.99 −2.16 8.45 1.75 −4.61 3.02 0.6
Causative Causative Colonizing Causative Causative Colonizing Colonizing Colonizing Colonizing Causative Causative Causative Causative Causative Causative Causative Causative Colonizing Causative Causative
22.15 23.48 24.22 23.25 24.25 22.63 23.47 25.29 23.88 24.38 25.69 24.91
24.91 23.67 24.16 23.85 26.23 22.36 22.88 25.21 24.5 23.8 25.25 24.64
Abbreviations of pathogens were S pne, Streptococcus pneumoniae; H inf, Haemophilus influenzae; M cat, Moraxella catarrhalis; P aer, Pseudomonas aeruginosa and K pne, Klebsiella pneumonia. ΔCt (Cthuman − Ctpathogen) represented the cut-off value to discriminate the pathogenic role from identified pathogens in the HIRA-TAN system, which was − 6 for S pne; −1 for H inf; −4 for P aer; −6 for K pne and 0 for M cat [RespMed2014]. Data were collected from 19 stored sputum samples.
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Table 4 Summary of the clinical study with pressure-based system equipped in the automated pipetting device. DNA extraction method
Centrifuge-based system
Pressure-based system
Overview of the devices Flow volume Sample volume Elution buffer PCR reaction solution Template DNA
– 200 μl 100 μl 24 μl 1 μl
30 ml/min 200 μl 500 μl 20 μl 5 μl
Results of clinical samples Cthuman DNA purity (260 nm/280 nm) Therapeutic target in commensal organisms Therapeutic target in foreign organisms Average time taken to perform STEP 5–15
24.05 ± 2.33 1.76 ± 0.11 29 2 1–2 h
24.11 ± 2.02 1.73 ± 0.14 29 (1.0) 2 (1.0) 0.5–1 h
Sputum was collected from patients who were diagnosed with pneumonia and analyzed as soon as expectorated. Data for Cthuman and DNA purity were mean ± SD. The concordance ratios for diagnosis of therapeutic target in commensal organisms and foreign organisms in HIRA-TAN system were shown in parentheses respectively (see Materials and methods). Data were collected from 50 sputum samples through clinical study.
filtration of the respiratory specimen at the spin column. As sputum viscosity was independent from gross appearance or DNA concentration [14], the sputum should be considered as a totally different specimen from other clinical samples. We coped with column clogging due to high viscosity by applying pressure twice whereby DNA was extractable. Despite the troubling aspect of sputum's viscosity, the pressurebased system could extract DNA and show proper Cthuman in all samples. Although, what caused small Cthuman from high viscosity samples needs to be further studied, the pressure system could be applied to the molecular diagnosis by using respiratory specimens. In conclusion, the pressure system was able to take the place of the centrifuge in a respiratory specimen and rapidly provide information to the bedside. Moreover, the pressure system yielded the space and time benefits of processing clinical samples and would promote the treatment of pneumonia by combining automatic pipetting device. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2015.03.023. Disclosure The authors have declared that no competing interests exist. This work was supported by the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (AS2321117F) from the Japan Science and Technology Agency and Grant-in-Aid for Young Scientists (B) (No. 23790918) from the Japan Society for the Promotion of Science. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. In addition, the manuscript has been reviewed by all of the authors and is not being
submitted to any other journals. All of the authors have declared that no competing interests exist. Acknowledgments For their collaboration and the enrollment of their patients, we are indebted to Makoto Nagata, MD, PhD and Minoru Kanazawa, MD, PhD, the Department of Respiratory Medicine, Saitama Medical University, Saitama and Etsuko Kishi, the Central Laboratory, Saitama Medical University, Saitama We would like to thank my colleague Johnathan Canton, PhD, Program in Cell Biology, Hospital for Sick Children, Toronto, Canada, for proofreading of manuscript draft. References [1] Hirama T, Yamaguchi T, Miyazawa H, et al. Prediction of the pathogens that are the cause of pneumonia by the battlefield hypothesis. PLoS One 2011;6:e24474. http://dx.doi.org/10.1371/journal.pone.0024474. [2] Hirama T, Minezaki S, Yamaguchi T, et al. HIRA-TAN: a real-time PCR-based system for the rapid identification of causative agents in pneumonia. Respir Med 2014;108: 395. http://dx.doi.org/10.1016/j.rmed.2013.11.018. [3] Wilson D, Yen-Lieberman B, Reischl U, Warshawsky I, Procop GW. Comparison of five methods for extraction of Legionella pneumophila from respiratory specimens. J Clin Microbiol 2004;42:5913–6. http://dx.doi.org/10.1128/JCM. 42.12.5913-5916. 2004. [4] Aldous WK, Pounder JI, Cloud JL, Woods GL. Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR. J Clin Microbiol 2005;43:2471–3. http://dx.doi.org/10.1128/JCM. 43.5. 2471-2473.2005. [5] Queipo-Ortuño MI, Tena F, Colmenero JD, Morata P. 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; 27:109–14. http://dx.doi.org/10.1007/s10096-007-0409-y. [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. http://dx.doi.org/10.1016/j.jviromet. 2010.10.024. [7] Deng J, Ma Z, Huang W, et al. Respiratory virus multiplex RT-PCR assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections. Virol Sin 2013;28:97–102. http://dx.doi.org/10.1007/s12250-013-3312-y. [8] Li J, Mao N-Y, Zhang C, et al. The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes. BMC Infect Dis 2012;12:189. http://dx.doi.org/10.1186/14712334-12-189. [9] Chotirmall SH, McElvaney NG. Fungi in the cystic fibrosis lung: bystanders or pathogens? Int J Biochem Cell Biol 2014;52:161–73. http://dx.doi.org/10.1016/j. biocel.2014.03.001. [10] Bouchara J-P, Hsieh HY, Croquefer S, et al. Development of an oligonucleotide array for direct detection of fungi in sputum samples from patients with cystic fibrosis. J Clin Microbiol 2009;47:142–52. http://dx.doi.org/10.1128/JCM. 01668-08. [11] Zhang Y, Zhu Y, Yao B, Fang Q. Nanolitre droplet array for real time reverse transcription polymerase chain reaction. Lab Chip 2011;11:1545–9. http://dx.doi.org/10. 1039/c0lc00502a. [12] Hwang K-Y, Kwon SH, Jung S-O, et al. Miniaturized bead-beating device to automate full DNA sample preparation processes for gram-positive bacteria. Lab Chip 2011;11: 3649–55. http://dx.doi.org/10.1039/c1lc20692c. [13] Lee AV, Atkinson C, Manuel RJ, Clark DA. Comparative evaluation of the QIAGEN QIAsymphony® SP system and bioMérieux NucliSens easyMAG automated extraction platforms in a clinical virology laboratory. J Clin Virol 2011;52:339–43. http://dx.doi.org/10.1016/j.jcv.2011.08.016. [14] Picot R, Das I, Reid L. Pus, deoxyribonucleic acid, and sputum viscosity. Thorax 1978; 33:235–42. http://dx.doi.org/10.1136/thx.33.2.235.
