Detection of Mycobacterium tuberculosis Using a Capillary

Subscriber access provided by EASTERN KENTUCKY UNIVERSITY

Article

Rapid detection of Mycobacterium tuberculosis using a capillaryarray microsystem with integrated DNA extraction, loopmediated isothermal amplification and fluorescence detection Dayu Liu, Guangtie Liang, Qiong Zhang, and Bin Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac400412m • Publication Date (Web): 22 Mar 2013 Downloaded from http://pubs.acs.org on March 26, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Rapid detection of Mycobacterium tuberculosis using a capillary-array microsystem with integrated DNA extraction, loop-mediated isothermal amplification and fluorescence detection Dayu Liu1,2*, Guangtie Liang1, Qiong Zhang1, Bin Chen1

1. Department of Laboratory Medicine, Guangzhou First Municipal People's Hospital Affiliated to Guangzhou Medical College 2. Laboratory of Clinical Chemical Technology, Department of Laboratory Medicine, the Second Affiliated Hospital of Dalian Medical University * Corresponding author. 1, Panfu Road, 510180, Guangzhou, China. Fax: 86-20-81048058; Tel: 86-20-81048027; E-mail: [email protected]

KEYWORDS : microfluidic system, integration, capillary array, loop-mediated isothermal amplification, tuberculosis

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 25

ABSTRACT

This paper reports the first time a highthroughput microfluidic system with fully integrated loopmediated isothermal amplification (LAMP) analysis. With the developed system, parallel Mycobacterium tuberculosis detections were implemented in polytetrafluoroethylene capillaries through the utilization of droplet technology coupled with magnetic beads. During the analysis, liquid plugs containing different types of sample or reagents are sequentially introduced into the capillaries and made to form droplets therein. The whole analytical process, including DNA extraction, LAMP and detection of the amplified products were conducted in such droplets. The developed microsystem is able to process 10 samples in parallel. The entire diagnostic procedure, from sample-in to answer-out, can be automatically completed within 50 minutes with a limit of detection (LOD) of ten bacteria. This microsystem was evaluated by analyzing clinical samples and clinical sensitivity (positive detection rate) of 96.8% and specificity (negative detection rate) of 100% was achieved. The presented capillary LAMP assay features highthroughput and low-cost, thus is a promising tool for rapid tuberculosis diagnosis.


2

Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Mycobacterium tuberculosis infection has been a persistent health problem, with 9.2 million cases reported per year in the world1. Its ease of transmission, ability to remain dormant in the bodies of apparently healthy people, and the lack of effective treatment present a unique challenge to public health authorities. Conventionally, laboratory procedures for clinical specimens involve microscopic examination for the presence of acid fast bacilli (AFB), and identification of the bacterium by culture. So far, both of these methods have limitations in providing efficient tuberculosis (TB) diagnosis: for microscopic examination, the proportion of positive AFB smears is only around 40-60%2; for bacterium culture examination, characterization of M. Tuberculosis infection requires about 4 weeks even in advanced laboratories, due to the extremely slow growth rate of the bacterium. Therefore, timely and reliable identification of M. Tuberculosis infection is needed for early treatment to improve prognosis and to reduce the risk of spreading to other hospitalized patients. In recent years, various kinds of molecular methods have been developed in attempt to overcome the shortcomings of the conventional TB diagnostics mentioned above, in particular, the detection of a pathogen-specific nucleic acid sequence3-5. A variety of gene amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA) and strand displacement amplification (SDA), have already been commercialized as kits and introduced into routine diagnostics6. Such approaches may allow rapid diagnosis with a degree of sensitivity and specificity comparable to or even better than that of classical culture methods. For example, realtime PCR techniques have been adopted as tools for the implementation of sensitive and accurate TB diagnosis in clinical laboratories. However, as the precision thermal cycling steps of PCR


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 25

require complex temperature control units and consume high amounts of energy, PCR systems are undesirable under resource-limited conditions. In comparison with PCR, the recently developed loop-mediated isothermal amplification (LAMP) technology7 is much more suitable for rapid diagnostic assay. The most significant advantage of the LAMP technique is its ability to amplify target nucleic acid sequences under isothermal conditions (60-65 ℃) , which offers the possibility of designing a miniature amplification system with low energy consumption. In addition, the final amplified stem-loop DNAs yields an amplification of 109 copies of target DNA molecules, so that approximately a 100-fold greater sensitivity for LAMP amplification is demonstrated when compared with a conventional PCR process. As a consequence, results of LAMP assay can be interpreted using a simplified optical system or even with the naked eyes. By taking advantage of this technology, LAMP has been applied to achieve rapid and costeffective diagnosis of infectious diseases8-9. Recently, miniature genetic analysis systems with integrated LAMP were also developed for the purpose of rapid detection of pathogenic agents. These µLAMP devices utilize the rapid thermal conduction from increased surface-to-volume ratio, as well as the low sample/reagent consumption attributable to reduced device sizes. Following this principle, a number of µLAMP approaches have been developed and introduced into different types of nucleic acid analyses, showing superiority over conventional apparatuses in terms of speed, miniaturization, power consumption, and cost10-18. Despite the attractiveness of these µLAMP assays, there are still some potential shortcomings in adapting them for clinical emergency disposal. Firstly, sample pre-treatment remains a technically demanding and timeconsuming step in these approaches. The quality of nucleic acid extraction always affects the results of genetic diagnosis. Consequently, well-trained personnel are still required for the laborintensive analytical process. Secondly, although there are few exceptions that reported µLAMP


4

Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


detection with integrated nucleic acid preparation13,15,17-18, such assays still face the limitation in analytical throughput and most of them can only process one sample at a time. Given that genetic diagnosis assays typically involve analysis of multiple samples, including target analyte(s), positive and negative controls, the “one by one” analytical mode would lead to an overall longer analysis time and increased chances of cross-contamination. Thirdly, most of the presented microfluidic devices require fabrication in specialized facilities, resulting in high production costs. These drawbacks described above severely limit the application of µLAMP assays in clinical diagnostics. Therefore, there is a great need to develop a complete “sample-in to answerout’’ LAMP assay by using a miniature system to carry out the entire protocol. More importantly, this assay must have a high throughput and a low running cost. To overcome the limitations associated with the current µLAMP devices, this paper reports a capillary-array microsystem, where DNA extraction, LAMP and online fluorescence detection can be organically integrated in arrayed capillaries by using the droplet technology coupled with usage of magnetic beads (MBs). The developed system has an inherent nature of small footprint size, thus allowing three functions of the TB diagnostic assay to be easily performed in parallel to achieve highthroughput detection. The entire procedure of TB diagnostic assay can be conducted in standard capillaries, resulting in reduced reaction volume and fabrication cost. To the best of our understanding, this is the first report of a microfluidic system that would permit the implementation of an integrated LAMP assay in parallel. With features of high sensitivity, function integrity and the ability to conduct parallel analysis, the developed microsystem could enable rapid, simple and highly efficient TB diagnosis.

2. Material and methods


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 25

2.1 Construction of the microsystem The µLAMP device (Fig.1a) was assembled with the following components: i) a heating block; ii) a multichannel syringe pump; iii) a bidirectional magnetic force controller, and iv) a fluorescence detection module. The heating block was constructed by assembling a grooved aluminum plate, Peltier heating element (Techsun, Beijing, China) and aluminum base support (Fig.1b). A PT-100 sensor (Sanxing, Dongyang, China) was embedded in the aluminum plates, provided temperature readings for feedback thermal control. The temperature signal was received by a computer through a USB interface, which then determined the power input to the Peltier heater using a fuzzy proportional-integral-derivative (PID) algorithm. Ten parallel grooves of 1.4 mm in width and 1.4 mm in depth were fabricated on the top surface of the aluminum plate. Standard polytetrafluoroethylene (PTFE) capillary tube (HongTai Silicon Fluorine Products Factory, Jiangsu, China) with an inner diameter of 1 mm, a wall thickness of 200 µm and a length of 50 cm was positioned into each of the grooves, capped with a polycarbonate cover sheet to enable close contact between capillaries and the grooves (Fig.1b). The capillary tubes were connected to a multichannel syringe pump (Longer Precision Pump Co. Ltd., Baoding, China), which facilitates the transportation of liquid sample to the tubes in parallel. The magnetic force controller (Landa Solenoid Factory, Zhongshan, Guangdong, China) contains two electromagnets, with a gap between them that allows traversing the capillary array (Fig.1c). Power input to the electromagnets was determined by the computer software. By modifying the power input, the two electromagnets can be temporarily kept in an all-off, oneon/one-off or alternatively switched on and off mode (10Hz) depending on the particular task.


6

Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


This permits the magnetic capturing, shaking and releasing of the MBs inside the capillaries become programmable. Droplet fluorescence excitation inside the parallel capillaries was achieved with a 480 nm bluelight light-emitting diode (LED) array (4 cm×4 cm), which is designed to illuminate nanogram amounts of DNA stains. The observing window for the compact detector, contains an amber emission filter plate fitting exactly over the blue excitation filter plate, with the capillary array positioned between these two filters (Fig.1d). The amber screen filters the blue excitation light, thereby allowing visual detection of emission light.

2.2 Sample collection and processing Bacillus Calmette Guerin (BCG) vaccine and sputum saliva samples were analyzed in this work. Samples serially diluted from the BCG vaccine (1×105 bacteria/ml, Huayang Biotech, Shanghai, China) with PBS were used as standards for LOD determination. During the operation, 100 µL of the dilution was added to a sterile plastic tube and then mixed with 200 µL of lysis buffer (1.25% SDS, 37.5% benzyl chloride, 10 mM Tris/HCl and 50 mM EDTA, pH 9)

19

. M.

tuberculosis(+) sputum saliva samples were obtained from Guangzhou Chest Hospital, China. All these clinical samples were verified by real-time PCR analysis. The sputum samples were collected in sterile plastic tubes, diluted to 1/2 of their initial concentrations with 5% trypsin (pH 8.0) prepared in 0.25 mmol/L CaCl2 solution. After being kept at room temperature for 30 minutes, 100 µL of the lysate was transferred to a sterile plastic tube and mixed with 200 µL of lysis buffer. Then, the plastic tube was vortexed and incubated at room temperature for 10 minutes. After further adding equal volume of binding buffer (Bioer, Hangzhou, China), the tube was thoroughly vortexed and the resultant mixture was ready for DNA extraction.


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 25

2.3 DNA extraction and LAMP-based DNA amplification in the capillary M. Tuberculosis DNA was extracted by using the droplet technology coupled with usage of MBs. Droplets containing the sample/reagent(s) were sequentially introduced into the capillary for DNA binding, MBs washing and DNA elution. In these steps, the MBs served as the solid carrier for DNA binding. The vibration of the MBs actuated by the twin electromagnets enhanced the interactions between the MBs and the solution inside a droplet, thus facilitating highly efficient MBs rinsing and DNA elution. Following the protocol reported in our previous work20, which comprises sample preparation for 15 min, DNA binding for 2 min, MBs rinsing twice for 4 min, and DNA elution at 50 °C for 1 min, the droplet-based DNA extraction can be completed in ~25 minutes. In this work, DNA extraction and amplification were coupled in a capillary. The coupled analytical process included several steps (Fig.2): (1) Six microliters of mineral oil (Amresco, Solon, Ohio), 2 µL of MB solution (Bioer, Hangzhou, China) and 25 µL of sample lysate were sequentially introduced into each capillary at a flow rate of 300 µL min-1. Such a plug was transported to pass through the magnetic field area, where the magnetic beads were trapped and then, the sample waste was flushed out; (2) Sample introduction and sample waste removing were repeated dozens of times to exhaust the sample lysate; (3) The MBs were then washed with 7.5× ThermoPol reaction Buffer (New England Biolabs, pH 8.9); (4) After the rinsing, a liquid train that was composed of 3 µL of H2O, 7 µL of mineral oil and 7 µL of LAMP reaction mixture (DEAOU Biotech, Guangzhou, China) was introduced into and advanced along the capillary; (5) Consequently, the DNA on MBs was eluted into the leading water plug; (6) When further


8

Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


advanced the liquid train along the capillary, the terminating plug (reaction buffer) caught up and merged with the leading one (eluted DNA). Therefore, the eluted DNA was mixed with the reaction buffer; (7) Eventually, the flow direction of the merged liquid plug was reversed, transporting it back to the heating block and maintained there at 65℃ for 30 minutes. It should be noted that, during DNA extraction (step 2 to 5), the MBs inside the aqueous phase were actuated up-and-down inside the capillaries (alternatively switched on and off mode, 10 Hz) to enhance the interaction between MBs and solutions.

2.4 Labeling and detection of LAMP products The amplified LAMP products were labeled with SYTO-81 DNA dyes (Life Technologies), which were added to the reaction mixture prior to the amplification. During the amplification, the reaction droplets were transported to the detection area every 5 minutes for fluorescence observing. Fluorescence photos of the capillary array were taken by a Nikon D5100 digital camera. Upon blue-light excitation, labelled LAMP products emitted light green fluorescence. A sample was judged to be M. Tuberculosis (+) on the presence of light green fluorescence in the reaction droplet.

3. Results and Discussion 3.1 Transportation of multi-phase liquid in a capillary This microfluidic assay involves the transportation of multi-phase liquid plugs in a PTFE capillary. It has been reported that the advancement of an oil-water plug in a PTFE capillary results in the formation of a water-in-oil droplet21 (a behavior determined by the spreading


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 25

parameters and relative surface areas of the oil and water22). Droplet formation in a PTFE capillary can be explained as followed: 1) The mineral oil wets the walls of the capillaries preferentially over the aqueous phase, due to the highly hydrophobic nature of the PTFE tube; 2) Due to the relative movement between these two phases, the aqueous phase has a higher mobility than that of the oil phase so it is gradually encapsulated by the latter; 3) Surface tension at the water-oil interface is higher than that at the oil-PTFE interface. Consequently, the aqueous phase does not come in contact with the walls as they are separated by a thin layer of oil. Conversely, when an oil phase plug approaches an aqueous phase plug, these two plugs move concurrently in the tube with an interface between them

23-24.

Under a more complicated condition, when a

water-oil-water plug was transported along a PTFE capillary, it was observed that: 1) the oil plug in the middle of the liquid train encapsulated the terminating water plug but not the leading one; 2) the terminating water plug moved faster than the oil plug and eventually merged with the leading one; and 3) when the liquid train was transported reversely, the merged aqueous-phase plug moved next to the oil plug and it was re-encapsulated by the oil (Fig.3 and supporting information 1). In brief, with interfacial chemistry, a water-in-oil droplet can be generated by allowing a liquid train of oil-water to flow along a PTFE capillary. The utilization of a droplet not only prevents evaporation during the thermal reaction but also eliminates the surface adsorption caused by interactions between the reaction components and the inner surface. Based on droplet generation in the capillary, sample or reagents can be loaded into a capillary sequentially without any crosscontamination. Therefore, all the steps related to the genetic diagnostic assay can be continuously implemented in a capillary.


10

Page 11 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.2 Droplet-based DNA extraction in a capillary The MB-based DNA extraction methods consist of a bind-wash-elute protocol25: The DNA adsorbs to the MBs under a low pH, high ionic strength environment, and is then eluted in a lowsalt, high pH solution. Therefore, the high-salinity LAMP reaction buffer can not be used directly to elute the DNA on MBs. In this work, a 7.5× ThermoPol buffer was used for MBs rinsing. Usage of this highly saline solution avoided loss of DNA during the rinsing step. After removing impurities from the MBs, a small quantity of ThermoPol buffer was still left on the MBs (volume of buffer left was determined to be ~0.1 µL per 2 µL MBs by comparing the difference in weight between dried and wetted MBs). When a water plug was transported to the magnet area, the small amount of ThermoPol buffer on the MBs was released into this plug (3 µL H2O). The resultant weakly alkaline buffer with a low ionic strength (~0.2× ThermoPol buffer, pH 8.9) is well suited to DNA elution. It is worthy of mention that aggregation of the MBs was observed while processing the sputum samples. This phenomenon may be due to the interactions between the impurities adsorbed on the surface of MBs, such nonspecific adsorption may affect the DNA extraction efficiency.

3.3 Coupling of DNA elution and LAMP in a capillary The coupling of DNA extraction and LAMP is most important aspect of the capillary LAMP assay. In the present work, the coupling of these two functions was achieved by partitioning and merging two aqueous-phase plugs that separated by an oil plug. As shown in Fig.2, after MB rinsing, a liquid train containing a section of oil capped by aqueous-phase plugs at either end was introduced into the capillary. Upon arrival at the MB area, the DNA on the MBs was firstly eluted in the leading aqueous-phase plug (sterile H2O). While the liquid train advanced further,


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 25

the terminating aqueous-phase plug (LAMP reaction mixture) moved to the front of the oil plug and consequently, merged with the leading aqueous-phase plug. Therefore, the eluted DNA was added to the reaction mixture. The merging instant can be controlled by setting the volume of mineral oil. In the present work, the volume of mineral oil used was 7 µL, and it was observed that the leading and the terminating plugs merged across a ~18 cm migration distance. When it was reversely transported, the liquid plug returned to the heating area and the merged aqueousphase plug was re-encapsulated by the oil phase due to its higher migration rate relative to the oil plug. Therefore, DNA extraction and LAMP were coupled in a capillary.

3.4 LAMP products detection in the capillary-array As DNA production efficiency of LAMP is remarkably higher than that of PCR, it is possible to detect the amplified products by observing the fluorescence of the intercalating DNA dyes. Due to the large amount of LAMP products, a higher concentration of fluorescence dye is needed. In this work, SYTO-81 was selected as the dye for fluorogenic LAMP detection since it is only marginally inhibitory to LAMP up to high concentrations26. It was found that the fluorescence in M. Tuberculosis (+) reaction droplets increased quickly with the extension of amplification time. Upon blue-light excitation, the M. Tuberculosis positive samples appeared light green in color while the M. Tuberculosis (-) ones only showed very weak fluorescence (Fig.4). The results demonstrated that a 25-minute reaction time was sufficient for identifying a M. Tuberculosis (+) sample. Within such a period of time, obvious amplification of the fluorescence intensity can be observed in droplets corresponding to BCG inputs ranging from 10 to 1×104. By observing the fluorescence signal with the naked eyes, it was possible to discriminate positive amplifications from negative ones.


12

Page 13 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.5 Detection limit and specificity of the TB diagnostic assay To demonstrate the detection limit of the µLAMP assay, serially diluted BCG suspensions were prepared and tested. The integrated analysis showed a limit-of-detection (LOD) of 10 bacteria (Fig.4). The high sensitivity of the µLAMP analysis is possibly attributable to the merits of loopmediated mechanism of the amplification and the sensitive fluorescence detection approach. To validate the specificity of the µLAMP assay, irrelevant bacteria not targeted by the LAMP primers, namely, Diplococcus pneumoniae, E. coli, Staphylococcus aureus and Klebsiella pneumoniae, were used as controls. In parallel, four randomly selected M. Tuberculosis (+) clinical samples were also tested. M. Tuberculosis DNA was extracted in the capillaries and was then subjected to LAMP amplifications. Fluorescence imaging of the droplets showed that the µLAMP assay was highly specific and did not bring about cross-reactions from non-targeted pathogens (Fig.5).

3.6 Evaluation of the μLAMP assay with clinical samples The µLAMP assay was applied to analyze clinical specimens, including 32 M. Tuberculosis(+) and 10 M. Tuberculosis(-) sputum samples, to test its feasibility in TB diagnosis. Among the 32 M. tuberculosis(+) sputum samples, 31 cases were identified as positive by using the µLAMP assay. The only one case that failed to be detected has a low level of M. Tuberculosis loading, with a DNA reading of 5×102 copy/mL. The failure in detecting this case can be ascribed to the limitation in DNA extraction from low-level M. Tuberculosis samples. As it has been demonstrated, the unspecific binding of impurities on MBs may affect DNA capturing, and accordingly lead to lower detection sensitivity. The 10 M. Tuberculosis(-) samples were analyzed


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 25

in parallel and showed no fluorescence. Figure of representative TB patient sample results is provided in supporting information 2. These results indicated that the developed µLAMP approach had the potential to be used in clinical TB diagnostics. Table 1 shows a comparison between the present µLAMP assay and the commercial TB diagnostics. The most important feature of the present method lies in its function integration, namely incorporating all the steps needed for M. Tuberculosis detection in a capillary. The integrated LAMP assay requires only ~40% of the analysis time (50 min vs. 120 min) needed by conventional methods and the reaction volume is reduced by 5-fold (10 µL vs. 50 µL). The possibility of conducting miniature genetic assays in parallel not only reduces the analysis time, but also simplifies the operation. Although the µLAMP is inferior to the conventional methods in terms of the number of test per run, its ability to run 10 tests in parallel is believed to be sufficient for on-the-spot analysis. With advantages in sensitivity, throughput and analysis time, the microsystem presented here is well suited to clinical practice.

Conclusions In this study, we developed a capillary-array microsystem for rapid genetic diagnosis of M. Tuberculosis. By using a protocol combining droplet and MBs, the entire process of TB diagnosis, from sample-in to answer-out, was integrated in a same device. This capillary-array system demonstrates three features: (1) the integration of all steps related to genetic detection of TB into a miniaturized device; (2) the capacity for parallel sample processing; and (3) low cost fabrication and analysis process. A limitation associated with the present method is its limited detection sensitivity in analyzing low-abundance M. Tuberculosis samples. To overcome this drawback, we aim to improve DNA extraction efficiency in our future work by increasing


14

Page 15 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


sample loading volume or applying an affinity-based M. Tuberculosis enrichment strategy. This system will eventually facilitate the realization of highly engineered equipment to perform assays for agents that are associated with epidemics.


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 25

FIGURES Figure 1

a. Schematic representation of the microsystem. 1. Syringe pump; 2. Heating block; 3. Bidirectional electromagnet; 4. Signal detection module; b. Schematic of the heating block. From top to bottom are: PC cover, capillary array, grooved aluminum plate (with embedded PT100 sensor) and the aluminum plate holder; c. The capillary array was sandwiched between two electromagnets. By modifying the power input, the two electromagnets can be temporarily kept in an all-off, one-on/one-off or alternatively switched on and off mode; d. Schematic of the signal detection module. From top to bottom are emission filter, capillary array, excitation filter and LED array.


16

Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2

Schematic of the processes of the µLAMP assay for M. Tuberculosis detection. In principle, the integrated µLAMP assay is conducted as follows: 1.Sample/reagents injection and DNA binding; 2. MBs immobilization; 3.MBs washing; 4. Introducing a water-oil-water plug into the capillary; 5.Elution of DNA into the leading water plug; 6. DNA release into LAMP reaction buffer by merging the leading and the terminating plugs; 7.Transportation of the merged droplet backward to the heating area for LAMP reaction; 8. Transportation of the LAMP droplet to the detection area, where the droplet fluorescence was excited with a blue-light LED array.


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 25

Figure 3

Continuous photos showing the transportation of a water-oil-water plug in a PTFE capillary. The leading water plug contained 3 µL of H2O (light blue) and the terminating water plug contained 7 µL of 1× ThermoPol reaction buffer (dark blue). The two plugs were separated by an oil plug (red). a. A water-oil-water plug was introduced into a capillary; b. During moving along the capillary, the oil plug in the middle of the liquid train encapsulated the terminating water plug; c. The terminating water plug moved faster than the oil plug and merged with the leading one after migrated for ~18 cm distance; d. when the liquid train was transported reversely, the merged aqueous-phase plug was re-encapsulated by the oil.


18

Page 19 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4

Investigation of limit-of-detection of the µLAMP assay. Capillaries from top to bottom corresponded to BCG inputs of 5, 101, 102, 103, 104 and 0 (negative control), respectively. Fluorescence photos were taken every 5 minutes.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 25

Figure 5

Fluorescence of the LAMP reaction droplets under blue-light excitation. Capillaries from top to bottom corresponded: 1. Diplococcus pneumonia, 2. E. coli, 3.Staphylococcus aureus, 4.Klebsiella pneumonia DNA; 5-8. Four randomly selected M. Tuberculosis(+) clinical samples.


20

Page 21 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. A comparison between the present µLAMP assay and the commercial methods for TB diagnosis

Present

Commercial

Commercial

µLAMP assay

LAMP

Real-time PCR

Analysis Time

~50 min

~120 min

~120 min

Reaction Volume

10 µL

20-50 µL

20-50 µL

Function Integration

Yes

No

No

Test per run

10

16–96

16–384

Cost

Low

Low

High

Portability

High

Low

Low

Supporting Information. 1. Continuous observing the transportation of a water-oil-water plug in a PTFE capillary. The leading water plug contained 3 µL of H2O (light blue) and the terminating water plug contained 7 µL of 1× ThermoPol reaction buffer (dark blue). These two plugs were separated by an oil plug (red). The video shows that during moving along the capillary, the oil plug in the middle of the liquid train encapsulated the terminating water plug. After moving a distance of ~18 cm, the terminating water plug merged with the leading one. When the flow direction of the liquid plug was reversed, the merged aqueous-phase plug was reencapsulated by oil. 2. Representative TB patient sample results obtained from the µLAMP assay


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

AUTHOR INFORMATION Corresponding Author *Dayu Liu *1, Panfu Road, 510180, Guangzhou, China. Fax: 86-20-81048058; Tel: 86-20-81048027; Email: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources National Natural Science Foundation of China (NSFC, No. 81171418, 812717 and 81201163), Science and Technology Bureau of Guangdong Province (No.2011B060100002), Guangzhou Municipal Science and Technology Bureau (No.2012J4100030), and Health Bureau of Guangzhou (No. 20121A021002 and 20121A011035).


22

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGMENT This study was sponsored by the National Natural Science Foundation of China (NSFC, No. 81171418, 812717 and 81201163), Science and Technology Bureau of Guangdong Province (No.2011B060100002), Guangzhou Municipal Science and Technology Bureau (No.2012J4100030), and Health Bureau of Guangzhou (No. 20121A021002 and 20121A011035). The authors gratefully acknowledge their financial supports. We thank Mr. Yaoju Tan at the Guangzhou Chest Hospital for his help in collecting tuberculosis specimens. We also thank Danny T.T. CHEUNG at Hong Kong University of Science and Technology for his language help. ABBREVIATIONS TB, Tuberculosis; LAMP, loop-mediated isothermal amplification; PTFE, polytetrafluoroethylene.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 25

REFERENCES (1) World-Health-Organization; WHO, Geneva, Switzerland., 2011. (2) Sarmiento, O. L.; Weigle, K. A.; Alexander, J.; Weber, D. J.; Miller, W. C. Journal of clinical microbiology 2003, 41, 3233-40. (3) Eichbaum, Q.; Rubin, E. J. American journal of clinical pathology 2002, 118 Suppl, S3-17. (4) Palomino, J. C. FEMS immunology and medical microbiology 2009, 56, 103-11. (5) Sankar, S.; Ramamurthy, M.; Nandagopal, B.; Sridharan, G. Molecular diagnosis & therapy 2011, 15, 1-11. (6) Versalovic, J.; Lupski, J. R. Trends in microbiology 2002, 10, S15-21. (7) Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N.; Hase, T. Nucleic acids research 2000, 28, E63. (8) Francois, P.; Tangomo, M.; Hibbs, J.; Bonetti, E. J.; Boehme, C. C.; Notomi, T.; Perkins, M. D.; Schrenzel, J. FEMS immunology and medical microbiology 2011, 62, 41-8. (9) Mori, Y.; Notomi, T. Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy 2009, 15, 62-9. (10) Wu, Q.; Jin, W.; Zhou, C.; Han, S.; Yang, W.; Zhu, Q.; Jin, Q.; Mu, Y. Analytical chemistry 2011, 83, 3336-42. (11) Ahmad, F.; Seyrig, G.; Tourlousse, D. M.; Stedtfeld, R. D.; Tiedje, J. M.; Hashsham, S. A. Biomedical microdevices 2011, 13, 929-37. (12) Hsieh, K.; Patterson, A. S.; Ferguson, B. S.; Plaxco, K. W.; Soh, H. T. Angew Chem Int Ed Engl 2012, 51, 4896-900. (13) Fang, X.; Chen, H.; Xu, L.; Jiang, X.; Wu, W.; Kong, J. Lab on a chip 2012, 12, 1495-9. (14) Fang, X.; Chen, H.; Yu, S.; Jiang, X.; Kong, J. Analytical chemistry 2011, 83, 690-5. (15) Fang, X.; Liu, Y.; Kong, J.; Jiang, X. Analytical chemistry 2010, 82, 3002-6. (16) Safavieh, M.; Ahmed, M. U.; Tolba, M.; Zourob, M. Biosensors & bioelectronics 2012, 31, 523-8. (17) Wang, C. H.; Lien, K. Y.; Wang, T. Y.; Chen, T. Y.; Lee, G. B. Biosensors & bioelectronics 2011, 26, 2045-52. (18) Wang, C. H.; Lien, K. Y.; Wu, J. J.; Lee, G. B. Lab on a chip 2011, 11, 1521-31. (19) Zhu, H.; Qu, F.; Zhu, L. H. Nucleic acids research 1993, 21, 5279-5280. (20) Liang, G. T.; Wang, Q. P.; Wang, W.; Liu, D. Y.; Zhou, X. M. Chinese J Anal Chem 2011, 39, 670-674. (21) Liu, D.; Liang, G.; Lei, X.; Chen, B.; Wang, W.; Zhou, X. Anal Chim Acta 2012, 718, 58-63. (22) Gennes, P. G. d.; Brochard-Wyart, F.; Quéré, D. Capillarity and Wetting Phenomena; Springer: Berlin, 2003. (23) Bico, J.; Quéré, D. Europhysics Letters 2000, 51, 546-550. (24) Bico, J.; Quéré, D. J. Fluid Mech. 2002, 467, 101-127. (25) Wen, J.; Legendre, L. A.; Bienvenue, J. M.; Landers, J. P. Analytical chemistry 2008, 80, 6472-9. (26) Stedtfeld, R. D.; Tourlousse, D. M.; Seyrig, G.; Stedtfeld, T. M.; Kronlein, M.; Price, S.; Ahmad, F.; Gulari, E.; Tiedje, J. M.; Hashsham, S. A. Lab on a chip 2012, 12, 1454-


24

Page 25 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


62. For TOC only


25

Detection of Mycobacterium tuberculosis Using a Capillary

Recommend Documents