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Thermal lysis and isothermal amplification of Mycobacterium tuberculosis H37Rv in one tube
Journal of Microbiological Methods 143 (2017) 1–5
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Thermal lysis and isothermal amplification of Mycobacterium tuberculosis H37Rv in one tube
MARK
Prasad Shettya,1, Dipayan Ghosha,1, Debjani Paula,b,⁎ a b
Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India Wadhwani Research Centre for Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Tuberculosis Isothermal amplification Helicase dependent amplification Sample preparation Disinfection One-step
Tuberculosis (TB) is a leading cause of high mortality rates in developing countries. Sample preparation is one of the major challenges in developing an inexpensive point-of-care device for rapid and confirmed detection of tuberculosis. Existing chemical and mechanical lysis methods are unsuitable for field applications, as they require intermediate wash steps, manual intervention or separate lysis equipment. We report a one-step reaction protocol (65 °C and 60 min) for the H37Rv strain of Mycobacterium tuberculosis that (i) completely disinfects the mycobacteria culture, (ii) lyses the cells and (iii) performs helicase dependent amplification on the extracted DNA. Our assay combines multiple functions in a single step, uses a dry heat bath and does not require any intermediate user intervention, which makes it suitable for use by minimally trained health workers at the point of care.
1. Introduction Tuberculosis (TB) is a communicable disease and a global health threat, especially in the developing countries. TB is caused by a bacterial pathogen Mycobacterium tuberculosis. Nucleic acid amplification tests (NAATs) for diagnosing TB are rapid, specific and sensitive (~ 86%) (Dinnes et al., 2007). But commercial NAAT platforms are not suitable for use in primary healthcare centres due to their high price, lack of robustness and need for trained personnel to operate. Therefore, the WHO has declared a need for nucleic acid-based tests on sputum samples that are suitable for use in microscopy centres (WHO, 2014). Most of the NAAT platforms rely on the polymerase chain reaction (PCR) to amplify the nucleic acids. Unlike PCR, isothermal amplification techniques, such as, helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), loop-mediated amplification (LAMP), etc. can amplify the nucleic acid at a single incubation temperature (Gill and Ghaemi, 2008). These techniques can be performed with low-cost heat sources, such as, hot plates, heat blocks and hand/toe warmers (Huang et al., 2013; Shetty et al., 2016). Recently we reported rapid (~ 10 min) helicase dependent amplification of M. tuberculosis (H37Rv) DNA on a disposable paper substrate (Shetty et al., 2016). We performed off-chip sample preparation prior to amplifying the DNA on the paper chip. In most lab protocols, sample preparation involves two primary processes: (a) disinfection (i.e.
⁎
1
making the pathogen in sputum/culture samples non-viable) or decontamination (i.e. suppressing the growth of other bacteria in the sample to ensure M. tuberculosis growth) and, (b) lysis (e.g. DNA extraction and purification). Disinfection (Best et al., 1988; Rikimaru et al., 2002) or decontamination (Helb et al., 2010) of mycobacteria is done chemically, while lysis is performed chemically, mechanically or thermally. As chemical lysis requires a number of centrifugation steps and manual washes, it is difficult to integrate into a point-of-care device. Existing mechanical lysis techniques (Ferguson et al., 2016) also require frequent manual intervention. The advantage of thermal lysis is that it does not require incompatible chemicals, or separate equipment. Thermal lysis of M. tuberculosis at 80 °C (Doig et al., 2002; Warren et al., 2006) has been demonstrated. The high temperatures (95 °C) employed during PCR (Elbir et al., 2008) can also release DNA directly from cells. Some groups have amplified tuberculosis DNA by isothermal LAMP assays (Li et al., 2014; Rudeeaneksin et al., 2012) after performing DNA extraction separately. Another group (Bentaleb et al., 2016) demonstrated LAMP on sputum samples after chemically decontaminating the sputum is a separate step prior to amplification. Here, we have combined the three steps of disinfection, lysis and helicase dependent amplification (HDA) of M. tuberculosis into a single heat incubation step performed at 65 °C. All experiments in this study were performed using the standard H37Rv laboratory strain of M.
Corresponding author. E-mail address: [email protected] (D. Paul). Equal contributions.
http://dx.doi.org/10.1016/j.mimet.2017.09.013 Received 3 August 2017; Received in revised form 16 September 2017; Accepted 16 September 2017 Available online 19 September 2017 0167-7012/ © 2017 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods 143 (2017) 1–5
P. Shetty et al.
is given in the supplementary information.
tuberculosis. The complete assay takes place in a single tube requiring no intermediate intervention. The helicase dependent amplification protocol amplified DNA in presence of cell debris. There was no colony growth from the thermal lysate, which confirmed complete disinfection. To the best of our knowledge, this is the first demonstration of complete disinfection, lysis and helicase dependent amplification of M. tuberculosis H37Rv strain achieved in a single heat incubation step at 65 °C. Our one-step protocol (a) renders the mycobacteria non-viable, (b) functions without any user intervention, (c) does not require any additional equipment, and (d) can be safely performed in low-resource settings with limited biosafety measures.
To perform the combined disinfection and thermal lysis protocol, 100 μl of the live liquid culture (containing typically 107 CFU/ml) was transferred to a 200 μl thin-walled micro-centrifuge tube and kept on a heat block. We tested the protocol at different temperatures (95 °C and 65 °C) and for different durations (60 min and 30 min). The thermal lysate was directly used as the template for HDA without any purification.
2. Materials and methods
2.5. Checking cell viability after heat disinfection
2.1. Equipment and chemicals
For checking the viability of the lysed cells, 10 μl of M. tuberculosis lysate was plated on M7H11 agar plates supplemented with oleic albumin dextrose catalase (OADC). The plates were incubated up to 8 weeks at 37 °C and monitored regularly for any signs of cell viability. Cell viability after thermal disinfection and lysis was checked by performing four separate experiments. Each experiment was performed with duplicate plates. Images of the bacterial plates were acquired after 3 or 4 weeks for comparison of the various lysates with the unlysed sample.
2.4. Combined heat disinfection and thermal lysis conditions
We used a MJ Mini thermal cycler from Bio-Rad (India) for PCR and a temperature-controlled heat block from Trishul Equipment (India) for isothermal amplification. FastPrep 24 mechanical lysis instrument and kit were bought from MP Biomedicals (USA). PureLyse kit from Claremont BioSolutions (USA) was also used for mechanical lysis. The concentration of DNA was measured using a NanoDrop instrument (NanoPhotometer® P 300, IMPLEN) (Germany). The electrophoresis unit was bought from Genetix (India). PerkinElmer Geliance 1000 imaging system (India) was used for imaging the agarose gels. M7H9 Middlebrook, albumin dextrose catalase (ADC) and Luria agar for growing the mycobacteria were bought from Himedia (India). Middlebrook 7H11 agar base, glycerol, and Tween 80 were purchased from Sigma Aldrich (India). Middlebrook oleic albumin dextrose catalase (OADC) enrichment medium was obtained from Beckton Dickinson (India). Chemical DNA extraction was performed with DNAeasy Blood and tissue kit, purchased from Qiagen (India). IsoAmp III enzyme mix for helicase dependent amplification was purchased from Biohelix Inc. (Beverly, USA). We got the amplification primers synthesized by Integrated DNA Technologies (India). Deoxyribonucleotide triphosphate mix (dNTP) and deoxyadenosine triphosphate (dATP) were purchased from New England Biolabs (India). Filter tips (ART barrier specialty pipette tips), ethidium bromide and DNA ladder (O'GeneRuler ultra low range 10–300 bp ladder) were obtained from Thermo Scientific (India). The M. tuberculosis strain (H37Rv) was kindly donated by the Foundation for Medical Research, Mumbai.
2.6. Helicase dependent amplification After each lysis experiment, helicase dependent amplification was performed at 65 °C using 10 μl reaction volume. Either the lysate or the purified DNA (from chemical and mechanical lyses) was used as the template for HDA for initial optimization. An 84 bp region of the IS6110 gene was targeted using a previously reported primer set (Motré et al., 2011). The IsoAmp III enzyme mix includes T4 gene 32 protein, exo-klenow fragment of DNA polymerase I, E. coli UvrD helicase, and the accessory protein MutL. We used ~2.5 × concentration of the enzyme mix for all our reactions. The remaining reaction components were 1× annealing buffer II, 4 mM MgSO4, 40 mM NaCl, 0.4 mM of each dNTP, 4.65 mM additional dATP, forward and reverse primers (0.12 μM each), DNA template (either purified genomic DNA or whole bacterial cells) and DNase-free water. The amplified DNA was detected in a 4% agarose gel stained with ethidium bromide (0.5 μg/ml). The mean greyscale pixel intensities of the gel bands corresponding to pure DNA (obtained by chemical or mechanical lysis) and the one-step reaction were obtained using the image analysis software ImageJ.
2.2. Growth of M. tuberculosis (H37Rv) All work with M. tuberculosis H37Rv was performed in the biosafety level 3 (BSL-3) facility of the Foundation for Medical Research, Mumbai. M. tuberculosis was cultured on M7H9 Middlebrook liquid media. The final broth was prepared by mixing 0.52 g M7H9, 440 μl of glycerol, 150 μl of Tween 80 and 100 ml of water. The broth was then autoclaved at 121 °C for 20 min. 10% v/v filtered albumin dextrose catalase (ADC) was added into the media (i.e. 1 ml of ADC in 9 ml of M7H9 liquid media) after it cooled down to 40 °C. Then 1% inoculum (i.e. 0.1 ml inoculum in 10 ml liquid media approximating to 107–108 CFU/ml) was added to the broth and kept in an incubator at 37 °C without any shaking to prevent aerosol formation in the incubator.
2.7. Single step protocol combining disinfection, thermal lysis and HDA For the combined thermal disinfection, lysis and amplification protocol (i.e. the schematic shown in Fig. 1), the cell culture was directly incubated along with the HDA reaction mixture. The HDA reaction mix was spiked with 2 μl of cultured H37Rv strain (107 CFU/ml) and heated at 65 °C for 60 min. This combined protocol is referred to as ‘one-step’ throughout the manuscript. We performed this experiment four times and each experiment was performed in duplicate. 3. Results and discussion
2.3. Chemical and mechanical lysis conditions
3.1. Validating the combined thermal disinfection and lysis protocol using H37Rv
Chemical lysis was performed according to the protocol accompanying the Qiagen DNAeasy blood and tissue kit for gram-positive bacteria. Mechanical lysis was performed using the recommended protocols of the FastPrep instrument or the PureLyse kit, both of which are based on bead beating. However, we did not perform any separate disinfection of the bacteria before carrying out any of the lysis protocols. The detailed description of the chemical and mechanical lysis steps
All reported protocols for thermal disinfection (or lysis) of M. tuberculosis involve high (> 80 °C) temperatures to render the pathogen inactive. Therefore, we also added a positive lysis control by incubating the sample at 95 °C for 30 min. Fig. 2 shows the cell viability after thermal disinfection and lysis. Panels (a) and (c) confirm that M. tuberculosis H37Rv can be successfully inactivated at 65 °C with no colony growth up to 3 weeks. We further monitored the plates for up to 2
Journal of Microbiological Methods 143 (2017) 1–5
P. Shetty et al.
Fig. 1. Schematic diagram of the combined disinfection, lysis and helicase dependent amplification (HDA) performed in a single step. The liquid culture of mycobacteria was added directly into the tube containing the amplification mixture and used as the template for HDA reaction. The reaction tubes were incubated at 65 °C for 60 min in a heat block. The amplified DNA was detected by gel electrophoresis.
(mechanical lysis) supports an earlier report (Ferguson et al., 2016) that recommends a separate chemical disinfection step. In case of chemical and thermal lysis, there was no bacterial growth after four weeks. These results confirm that heat incubation at a comparatively lower temperature of 65 °C for 30 min is as effective as chemical lysis in disinfecting the culture. This is an important achievement as combining the disinfection and the lysis steps into a single thermal reaction can make sample preparation much simpler. Chemical lysis involved repeated centrifugation steps for washing away the lysis reagents and took at least 1.5 h. In contrast, mechanical lysis using PureLyse took only 10 min. But there were several manual shearing steps and at least one instance of centrifugation. Thermal lysis did not involve any manual intervention. Based on its efficacy and
8 weeks (data not shown) and found that there were no colonies. Our observations confirm that there is no need for a separate chemical disinfection step when dealing with H37Rv cultures. Temperatures below 65 °C were not explored due to lack of compatibility with the HDA protocol.
3.2. Can different lysis techniques also disinfect H37Rv cultures? An important requirement of pathogenic sample preparation is to ensure that pathogens are inactivated. We explored whether standard chemical (Qiagen) and mechanical lysis (PureLyse) protocols are capable of disinfecting the M. tuberculosis culture. Fig. 3 shows the results of this comparison. The colony growth observed in case of PureLyse
Fig. 2. Viability of M. tuberculosis H37Rv strain after thermal lysis at a lower temperature (65 °C). These images were taken after 3 weeks. Panels (a) and (c) indicate plates containing the cells lysed for 30 min and 60 min respectively. Panel (b) shows the results of 30 min thermal lysis at the recommended temperature of 95 °C (positive control). As expected, the unlysed cells (negative control) in panel (d) show significant colony growth in just three weeks.
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Journal of Microbiological Methods 143 (2017) 1–5
P. Shetty et al.
Fig. 3. Effect of different lysis techniques on cell viability of M. tuberculosis (H37Rv strain) after 4 weeks of culture. Panel (a) indicates unlysed viable cells (negative lysis control). Panel (b) confirms that there are no colonies after chemical lysis. Panel (c) indicates that there is some colony growth after mechanical lysis. Panel (d) confirms that our thermal lysis protocol can disinfect the samples as effectively as chemical lysis.
another 60 min. This was done to establish whether the presence of debris in the lysate inhibits the HDA reaction. Finally, the live bacterial cells and the amplification mixture were added together into the reaction tube and the tube was incubated at 65 °C for 60 min. This was designated as ‘one-step’ reaction in the gel image (lanes 9 and 10). Successful and comparable amplification efficiency was observed for all lysis protocols. The greyscale pixel intensity of the gel bands obtained with pure DNA was 53.36 ± 8.11 (mean ± standard deviation over four experiments). The greyscale pixel intensity of the gel bands obtained from one-step reaction was 57.76 ± 6.89 (mean ± standard deviation over four experiments). These intensity values indicate that the DNA yield from our protocol was comparable to that obtained with pure DNA. Our results confirm that it is indeed possible to integrate disinfection, lysis and HDA of M. tuberculosis (H37Rv strain) into a single heat incubation step at 65 °C for 60 min, thereby simplifying the sample preparation.
simplicity, thermal lysis at 65 °C is well suited for integration into an automated diagnostic platform. 3.3. Can we achieve disinfection, lysis and HDA of H37Rv in a single heat incubation reaction? Since our final aim was to design an affordable, automated and simple diagnostic assay for NAAT-based detection of M. tuberculosis at the point of care, we explored whether the requirements of sample disinfection, cell lysis and DNA amplification could be combined into a single heat incubation step at 65 °C for 60 min. All reactants would be added into the tube before the one-step reaction, so that no user intervention is required. Fig. 4 shows the results of the combined disinfection, lysis and HDA of the IS6110 gene of M. tuberculosis (H37Rv strain). In positive controls (lanes 4 and 5), we used purified genomic DNA (obtained from mechanical and chemical lysis respectively) as the starting material for HDA (60 min). We also performed HDA directly on the lysates obtained from mechanical (lane 6) and thermal lyses (lanes 7 and 8). Thermal lysis was performed for 30 min, before adding the HDA enzymes into the lysate and performing DNA amplification for
4. Conclusion Nucleic acid sample preparation is one of the most cumbersome steps for DNA-based diagnosis. We have addressed this problem for M. tuberculosis cultures (H37Rv) by successfully combining disinfection, thermal lysis and amplification into a single reaction step. Thermal lysis is best suited for integration into a point-of-care device as it is simple, and does not require user intervention or purification of DNA before amplification. The entire assay involves a heat incubation step (65 °C, 60 min) using a single reaction mix. We have demonstrated the proof of concept of this integrated sample preparation and DNA amplification protocol with a standard strain (H37Rv) of M. tuberculosis. Our next goal is validating the protocol with a large number of clinical sputum samples. Acknowledgements The study was supported by a seed grant (DGDON446) from the Wadhwani Research Centre for Bioengineering (WRCB), Indian Institute of Technology Bombay. The authors thank Dr. Nerges Mistry and the Foundation for Medical Research (Mumbai) for the H37Rv strain and providing access to the BSL3 facility. The authors would like to thank Kayzad Nilgiriwala, Akshata Papewar, Nithya Kalyani Ganesan, Rupali Kekani, Anirvan Chatterjee, Aparna Tripathi, Priyanka Naik and Akshi Gupta for technical help. They also acknowledge the members of the Microfluidics and Biological Physics group, Department of Biosciences and Bioengineering, IIT Bombay, for constructive criticism on the manuscript and interesting discussions.
Fig. 4. Thermal disinfection, lysis and helicase dependent amplification of M. tuberculosis (H37Rv) achieved in a single heat incubation step at 65 °C for 60 min. Lane 1: 10–300 bp DNA ladder. Lane 2: HDA reaction without any template DNA (negative control). Some non-specific primer-dimer formation can be seen though. Lane 3: HDA reaction in the absence of the amplification enzymes (negative control). Lanes 4 and 5: HDA of M. tuberculosis using purified genomic DNA as the starting material (positive controls). The DNA in lane 4 was extracted by mechanical lysis, whereas the DNA in lane 5 was obtained by chemical lysis. Lane 6: HDA using mechanically lysed M. tuberculosis (using FastPrep) cells as the starting material. HDA was performed on the lysate without DNA purification. Lanes 7–8: Results of two step thermal lysis and HDA of M. tuberculosis. Lanes 9–10: Results of one-step disinfection, thermal lysis and HDA of cultured M. tuberculosis. Lanes 7–10 shows results obtained using M. tuberculosis cells directly as starting material.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2017.09.013. 4
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Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. J. Clin. Microbiol. 48, 229–237. Huang, S., Do, J., Mahalanabis, M., Fan, A., Zhao, L., Jepeal, L., Singh, S.K., Klapperich, C.M., 2013. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis. PLoS One 8 (3), e60059. Li, Y., Shi, L., Pan, A., Cao, W., Chen, X., Meng, H., Yan, H., Miyoshi, S., Ye, L., 2014. Evaluation of real-time loop-mediated isothermal amplification (RealAmp) for rapid detection of Mycobacterium tuberculosis from sputum samples. J. Microbiol. Methods 104, 55–58. Motré, A., Kong, R., Li, Y., 2011. Improving isothermal DNA amplification speed for the rapid detection of Mycobacterium tuberculosis. J. Microbiol. Methods 84, 343–345. Rikimaru, T., Kondo, M., Kajimura, K., Hashimoto, K., Oyamada, K., Miyazaki, S., Sagawa, K., Aizawa, H., Oizumi, K., 2002. Efficacy of common antiseptics against multidrug-resistant Mycobacterium tuberculosis. Int. J. Tuberc. Lung Dis. 6, 763–770. Rudeeaneksin, J., Bunchoo, S., Srisungngam, S., Sawanpanyalert, P., Chamnangrom, S., Kamolwat, A., Thanasripakdeekul, P., Taniguchi, T., Nakajima, C., Suzuki, Y., Phetsuksiri, B., 2012. Rapid identification of Mycobacterium tuberculosis in BACTEC MGIT960 cultures by in-house loop-medicated isothermal amplification. Jpn. J. Infect. Dis. 65, 306–311. Shetty, P., Ghosh, D., Singh, M., Tripathi, A., Paul, D., 2016. Rapid amplification of Mycobacterium tuberculosis DNA on a paper substrate. RSC Adv. 6, 56205–56212. Warren, R., de Kock, M., Engelke, E., Myburgh, R., Gey van Pittius, N., Victor, T., van Helden, P., 2006. Safe Mycobacterium tuberculosis DNA extraction method that does not compromise integrity. J. Clin. Microbiol. 44 (1), 254–256. WHO, 2014. World Health Organization. High-priority Target Product Profiles for New Tuberculosis Diagnostics: Report of a Consensus Meeting. (WHO reference number: WHO/HTM/TB/2014.18 (http://apps.who.int/iris/bitstream/10665/135617/1/ WHO_HTM_TB_2014.18_eng.pdf?ua=1&ua=1. Accessed 02.08.2017)).
References Bentaleb, E.M., Abid, M., El Messaoudi, M.D., Lakssir, B., Ressami, E.M., Amzazi, S., Sefrioui, H., Ait Benhassou, H., 2016. Development and evaluation of an in-house single step loop-mediated isothermal amplification (SS-LAMP) assay for the detection of Mycobacterium tuberculosis complex in sputum samples from Moroccan patients. BMC Infect. Dis. 16 (1), 517. Best, M., Sattar, S.A., Springthorpe, V.S., Kennedy, M.E., 1988. Comparative mycobactericidal efficacy of chemical disinfectants in suspension and carrier tests. Appl. Environ. Microbiol. 54, 2856–2858. Dinnes, J., Deeks, J., Kunst, H., Gibson, A., Cummins, E., Waugh, N., Drobniewski, F., Lalvani, A., 2007. A systematic review of rapid diagnostic tests for the detection of tuberculosis infection. Health Technol. Assess. 11 (3), 1–196. Doig, C., Seagar, A.L., Watt, B., Forbes, K.J., 2002. The efficacy of the heat killing of Mycobacterium tuberculosis. J. Clin. Pathol. 55, 778–779. Elbir, H., Abdel-Muhsin, A.M., Babiker, A., 2008. A one-step DNA PCR-based method for the detection of Mycobacterium tuberculosis complex grown on Lowenstein-Jensen media. Am. J. Trop. Med. Hyg. 78 (2), 316–317. Ferguson, T.M., Weigel, K.M., Lakey Becker, A., Ontengco, D., Narita, M., Tolstorukov, I., Doebler, R., Cangelosi, G.A., Niemz, A., 2016. Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis. Sci. Rep. 6, 19541. Gill, P., Ghaemi, A., 2008. Nucleic acid isothermal amplification technologies: a review. Nucleosides Nucleotides Nucleic Acids 27 (3), 224–243. Helb, D., Jones, M., Story, E., Boehme, C., Wallace, E., Ho, K., Kop, J., Owens, M.R., Rodgers, R., Banada, P., Safi, H., Blakemore, R., Lan, N.T., Jones-López, E.C., Levi, M., Burday, M., Ayakaka, I., Mugerwa, R.D., McMillan, B., Winn-Deen, E., Christel, L., Dailey, P., Perkins, M.D., Persing, D.H., Alland, D., 2010. Rapid detection of
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Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Thermal lysis and isothermal amplification of Mycobacterium tuberculosis H37Rv in one tube
MARK
Prasad Shettya,1, Dipayan Ghosha,1, Debjani Paula,b,⁎ a b
Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India Wadhwani Research Centre for Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Tuberculosis Isothermal amplification Helicase dependent amplification Sample preparation Disinfection One-step
Tuberculosis (TB) is a leading cause of high mortality rates in developing countries. Sample preparation is one of the major challenges in developing an inexpensive point-of-care device for rapid and confirmed detection of tuberculosis. Existing chemical and mechanical lysis methods are unsuitable for field applications, as they require intermediate wash steps, manual intervention or separate lysis equipment. We report a one-step reaction protocol (65 °C and 60 min) for the H37Rv strain of Mycobacterium tuberculosis that (i) completely disinfects the mycobacteria culture, (ii) lyses the cells and (iii) performs helicase dependent amplification on the extracted DNA. Our assay combines multiple functions in a single step, uses a dry heat bath and does not require any intermediate user intervention, which makes it suitable for use by minimally trained health workers at the point of care.
1. Introduction Tuberculosis (TB) is a communicable disease and a global health threat, especially in the developing countries. TB is caused by a bacterial pathogen Mycobacterium tuberculosis. Nucleic acid amplification tests (NAATs) for diagnosing TB are rapid, specific and sensitive (~ 86%) (Dinnes et al., 2007). But commercial NAAT platforms are not suitable for use in primary healthcare centres due to their high price, lack of robustness and need for trained personnel to operate. Therefore, the WHO has declared a need for nucleic acid-based tests on sputum samples that are suitable for use in microscopy centres (WHO, 2014). Most of the NAAT platforms rely on the polymerase chain reaction (PCR) to amplify the nucleic acids. Unlike PCR, isothermal amplification techniques, such as, helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), loop-mediated amplification (LAMP), etc. can amplify the nucleic acid at a single incubation temperature (Gill and Ghaemi, 2008). These techniques can be performed with low-cost heat sources, such as, hot plates, heat blocks and hand/toe warmers (Huang et al., 2013; Shetty et al., 2016). Recently we reported rapid (~ 10 min) helicase dependent amplification of M. tuberculosis (H37Rv) DNA on a disposable paper substrate (Shetty et al., 2016). We performed off-chip sample preparation prior to amplifying the DNA on the paper chip. In most lab protocols, sample preparation involves two primary processes: (a) disinfection (i.e.
⁎
1
making the pathogen in sputum/culture samples non-viable) or decontamination (i.e. suppressing the growth of other bacteria in the sample to ensure M. tuberculosis growth) and, (b) lysis (e.g. DNA extraction and purification). Disinfection (Best et al., 1988; Rikimaru et al., 2002) or decontamination (Helb et al., 2010) of mycobacteria is done chemically, while lysis is performed chemically, mechanically or thermally. As chemical lysis requires a number of centrifugation steps and manual washes, it is difficult to integrate into a point-of-care device. Existing mechanical lysis techniques (Ferguson et al., 2016) also require frequent manual intervention. The advantage of thermal lysis is that it does not require incompatible chemicals, or separate equipment. Thermal lysis of M. tuberculosis at 80 °C (Doig et al., 2002; Warren et al., 2006) has been demonstrated. The high temperatures (95 °C) employed during PCR (Elbir et al., 2008) can also release DNA directly from cells. Some groups have amplified tuberculosis DNA by isothermal LAMP assays (Li et al., 2014; Rudeeaneksin et al., 2012) after performing DNA extraction separately. Another group (Bentaleb et al., 2016) demonstrated LAMP on sputum samples after chemically decontaminating the sputum is a separate step prior to amplification. Here, we have combined the three steps of disinfection, lysis and helicase dependent amplification (HDA) of M. tuberculosis into a single heat incubation step performed at 65 °C. All experiments in this study were performed using the standard H37Rv laboratory strain of M.
Corresponding author. E-mail address: [email protected] (D. Paul). Equal contributions.
http://dx.doi.org/10.1016/j.mimet.2017.09.013 Received 3 August 2017; Received in revised form 16 September 2017; Accepted 16 September 2017 Available online 19 September 2017 0167-7012/ © 2017 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods 143 (2017) 1–5
P. Shetty et al.
is given in the supplementary information.
tuberculosis. The complete assay takes place in a single tube requiring no intermediate intervention. The helicase dependent amplification protocol amplified DNA in presence of cell debris. There was no colony growth from the thermal lysate, which confirmed complete disinfection. To the best of our knowledge, this is the first demonstration of complete disinfection, lysis and helicase dependent amplification of M. tuberculosis H37Rv strain achieved in a single heat incubation step at 65 °C. Our one-step protocol (a) renders the mycobacteria non-viable, (b) functions without any user intervention, (c) does not require any additional equipment, and (d) can be safely performed in low-resource settings with limited biosafety measures.
To perform the combined disinfection and thermal lysis protocol, 100 μl of the live liquid culture (containing typically 107 CFU/ml) was transferred to a 200 μl thin-walled micro-centrifuge tube and kept on a heat block. We tested the protocol at different temperatures (95 °C and 65 °C) and for different durations (60 min and 30 min). The thermal lysate was directly used as the template for HDA without any purification.
2. Materials and methods
2.5. Checking cell viability after heat disinfection
2.1. Equipment and chemicals
For checking the viability of the lysed cells, 10 μl of M. tuberculosis lysate was plated on M7H11 agar plates supplemented with oleic albumin dextrose catalase (OADC). The plates were incubated up to 8 weeks at 37 °C and monitored regularly for any signs of cell viability. Cell viability after thermal disinfection and lysis was checked by performing four separate experiments. Each experiment was performed with duplicate plates. Images of the bacterial plates were acquired after 3 or 4 weeks for comparison of the various lysates with the unlysed sample.
2.4. Combined heat disinfection and thermal lysis conditions
We used a MJ Mini thermal cycler from Bio-Rad (India) for PCR and a temperature-controlled heat block from Trishul Equipment (India) for isothermal amplification. FastPrep 24 mechanical lysis instrument and kit were bought from MP Biomedicals (USA). PureLyse kit from Claremont BioSolutions (USA) was also used for mechanical lysis. The concentration of DNA was measured using a NanoDrop instrument (NanoPhotometer® P 300, IMPLEN) (Germany). The electrophoresis unit was bought from Genetix (India). PerkinElmer Geliance 1000 imaging system (India) was used for imaging the agarose gels. M7H9 Middlebrook, albumin dextrose catalase (ADC) and Luria agar for growing the mycobacteria were bought from Himedia (India). Middlebrook 7H11 agar base, glycerol, and Tween 80 were purchased from Sigma Aldrich (India). Middlebrook oleic albumin dextrose catalase (OADC) enrichment medium was obtained from Beckton Dickinson (India). Chemical DNA extraction was performed with DNAeasy Blood and tissue kit, purchased from Qiagen (India). IsoAmp III enzyme mix for helicase dependent amplification was purchased from Biohelix Inc. (Beverly, USA). We got the amplification primers synthesized by Integrated DNA Technologies (India). Deoxyribonucleotide triphosphate mix (dNTP) and deoxyadenosine triphosphate (dATP) were purchased from New England Biolabs (India). Filter tips (ART barrier specialty pipette tips), ethidium bromide and DNA ladder (O'GeneRuler ultra low range 10–300 bp ladder) were obtained from Thermo Scientific (India). The M. tuberculosis strain (H37Rv) was kindly donated by the Foundation for Medical Research, Mumbai.
2.6. Helicase dependent amplification After each lysis experiment, helicase dependent amplification was performed at 65 °C using 10 μl reaction volume. Either the lysate or the purified DNA (from chemical and mechanical lyses) was used as the template for HDA for initial optimization. An 84 bp region of the IS6110 gene was targeted using a previously reported primer set (Motré et al., 2011). The IsoAmp III enzyme mix includes T4 gene 32 protein, exo-klenow fragment of DNA polymerase I, E. coli UvrD helicase, and the accessory protein MutL. We used ~2.5 × concentration of the enzyme mix for all our reactions. The remaining reaction components were 1× annealing buffer II, 4 mM MgSO4, 40 mM NaCl, 0.4 mM of each dNTP, 4.65 mM additional dATP, forward and reverse primers (0.12 μM each), DNA template (either purified genomic DNA or whole bacterial cells) and DNase-free water. The amplified DNA was detected in a 4% agarose gel stained with ethidium bromide (0.5 μg/ml). The mean greyscale pixel intensities of the gel bands corresponding to pure DNA (obtained by chemical or mechanical lysis) and the one-step reaction were obtained using the image analysis software ImageJ.
2.2. Growth of M. tuberculosis (H37Rv) All work with M. tuberculosis H37Rv was performed in the biosafety level 3 (BSL-3) facility of the Foundation for Medical Research, Mumbai. M. tuberculosis was cultured on M7H9 Middlebrook liquid media. The final broth was prepared by mixing 0.52 g M7H9, 440 μl of glycerol, 150 μl of Tween 80 and 100 ml of water. The broth was then autoclaved at 121 °C for 20 min. 10% v/v filtered albumin dextrose catalase (ADC) was added into the media (i.e. 1 ml of ADC in 9 ml of M7H9 liquid media) after it cooled down to 40 °C. Then 1% inoculum (i.e. 0.1 ml inoculum in 10 ml liquid media approximating to 107–108 CFU/ml) was added to the broth and kept in an incubator at 37 °C without any shaking to prevent aerosol formation in the incubator.
2.7. Single step protocol combining disinfection, thermal lysis and HDA For the combined thermal disinfection, lysis and amplification protocol (i.e. the schematic shown in Fig. 1), the cell culture was directly incubated along with the HDA reaction mixture. The HDA reaction mix was spiked with 2 μl of cultured H37Rv strain (107 CFU/ml) and heated at 65 °C for 60 min. This combined protocol is referred to as ‘one-step’ throughout the manuscript. We performed this experiment four times and each experiment was performed in duplicate. 3. Results and discussion
2.3. Chemical and mechanical lysis conditions
3.1. Validating the combined thermal disinfection and lysis protocol using H37Rv
Chemical lysis was performed according to the protocol accompanying the Qiagen DNAeasy blood and tissue kit for gram-positive bacteria. Mechanical lysis was performed using the recommended protocols of the FastPrep instrument or the PureLyse kit, both of which are based on bead beating. However, we did not perform any separate disinfection of the bacteria before carrying out any of the lysis protocols. The detailed description of the chemical and mechanical lysis steps
All reported protocols for thermal disinfection (or lysis) of M. tuberculosis involve high (> 80 °C) temperatures to render the pathogen inactive. Therefore, we also added a positive lysis control by incubating the sample at 95 °C for 30 min. Fig. 2 shows the cell viability after thermal disinfection and lysis. Panels (a) and (c) confirm that M. tuberculosis H37Rv can be successfully inactivated at 65 °C with no colony growth up to 3 weeks. We further monitored the plates for up to 2
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Fig. 1. Schematic diagram of the combined disinfection, lysis and helicase dependent amplification (HDA) performed in a single step. The liquid culture of mycobacteria was added directly into the tube containing the amplification mixture and used as the template for HDA reaction. The reaction tubes were incubated at 65 °C for 60 min in a heat block. The amplified DNA was detected by gel electrophoresis.
(mechanical lysis) supports an earlier report (Ferguson et al., 2016) that recommends a separate chemical disinfection step. In case of chemical and thermal lysis, there was no bacterial growth after four weeks. These results confirm that heat incubation at a comparatively lower temperature of 65 °C for 30 min is as effective as chemical lysis in disinfecting the culture. This is an important achievement as combining the disinfection and the lysis steps into a single thermal reaction can make sample preparation much simpler. Chemical lysis involved repeated centrifugation steps for washing away the lysis reagents and took at least 1.5 h. In contrast, mechanical lysis using PureLyse took only 10 min. But there were several manual shearing steps and at least one instance of centrifugation. Thermal lysis did not involve any manual intervention. Based on its efficacy and
8 weeks (data not shown) and found that there were no colonies. Our observations confirm that there is no need for a separate chemical disinfection step when dealing with H37Rv cultures. Temperatures below 65 °C were not explored due to lack of compatibility with the HDA protocol.
3.2. Can different lysis techniques also disinfect H37Rv cultures? An important requirement of pathogenic sample preparation is to ensure that pathogens are inactivated. We explored whether standard chemical (Qiagen) and mechanical lysis (PureLyse) protocols are capable of disinfecting the M. tuberculosis culture. Fig. 3 shows the results of this comparison. The colony growth observed in case of PureLyse
Fig. 2. Viability of M. tuberculosis H37Rv strain after thermal lysis at a lower temperature (65 °C). These images were taken after 3 weeks. Panels (a) and (c) indicate plates containing the cells lysed for 30 min and 60 min respectively. Panel (b) shows the results of 30 min thermal lysis at the recommended temperature of 95 °C (positive control). As expected, the unlysed cells (negative control) in panel (d) show significant colony growth in just three weeks.
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Fig. 3. Effect of different lysis techniques on cell viability of M. tuberculosis (H37Rv strain) after 4 weeks of culture. Panel (a) indicates unlysed viable cells (negative lysis control). Panel (b) confirms that there are no colonies after chemical lysis. Panel (c) indicates that there is some colony growth after mechanical lysis. Panel (d) confirms that our thermal lysis protocol can disinfect the samples as effectively as chemical lysis.
another 60 min. This was done to establish whether the presence of debris in the lysate inhibits the HDA reaction. Finally, the live bacterial cells and the amplification mixture were added together into the reaction tube and the tube was incubated at 65 °C for 60 min. This was designated as ‘one-step’ reaction in the gel image (lanes 9 and 10). Successful and comparable amplification efficiency was observed for all lysis protocols. The greyscale pixel intensity of the gel bands obtained with pure DNA was 53.36 ± 8.11 (mean ± standard deviation over four experiments). The greyscale pixel intensity of the gel bands obtained from one-step reaction was 57.76 ± 6.89 (mean ± standard deviation over four experiments). These intensity values indicate that the DNA yield from our protocol was comparable to that obtained with pure DNA. Our results confirm that it is indeed possible to integrate disinfection, lysis and HDA of M. tuberculosis (H37Rv strain) into a single heat incubation step at 65 °C for 60 min, thereby simplifying the sample preparation.
simplicity, thermal lysis at 65 °C is well suited for integration into an automated diagnostic platform. 3.3. Can we achieve disinfection, lysis and HDA of H37Rv in a single heat incubation reaction? Since our final aim was to design an affordable, automated and simple diagnostic assay for NAAT-based detection of M. tuberculosis at the point of care, we explored whether the requirements of sample disinfection, cell lysis and DNA amplification could be combined into a single heat incubation step at 65 °C for 60 min. All reactants would be added into the tube before the one-step reaction, so that no user intervention is required. Fig. 4 shows the results of the combined disinfection, lysis and HDA of the IS6110 gene of M. tuberculosis (H37Rv strain). In positive controls (lanes 4 and 5), we used purified genomic DNA (obtained from mechanical and chemical lysis respectively) as the starting material for HDA (60 min). We also performed HDA directly on the lysates obtained from mechanical (lane 6) and thermal lyses (lanes 7 and 8). Thermal lysis was performed for 30 min, before adding the HDA enzymes into the lysate and performing DNA amplification for
4. Conclusion Nucleic acid sample preparation is one of the most cumbersome steps for DNA-based diagnosis. We have addressed this problem for M. tuberculosis cultures (H37Rv) by successfully combining disinfection, thermal lysis and amplification into a single reaction step. Thermal lysis is best suited for integration into a point-of-care device as it is simple, and does not require user intervention or purification of DNA before amplification. The entire assay involves a heat incubation step (65 °C, 60 min) using a single reaction mix. We have demonstrated the proof of concept of this integrated sample preparation and DNA amplification protocol with a standard strain (H37Rv) of M. tuberculosis. Our next goal is validating the protocol with a large number of clinical sputum samples. Acknowledgements The study was supported by a seed grant (DGDON446) from the Wadhwani Research Centre for Bioengineering (WRCB), Indian Institute of Technology Bombay. The authors thank Dr. Nerges Mistry and the Foundation for Medical Research (Mumbai) for the H37Rv strain and providing access to the BSL3 facility. The authors would like to thank Kayzad Nilgiriwala, Akshata Papewar, Nithya Kalyani Ganesan, Rupali Kekani, Anirvan Chatterjee, Aparna Tripathi, Priyanka Naik and Akshi Gupta for technical help. They also acknowledge the members of the Microfluidics and Biological Physics group, Department of Biosciences and Bioengineering, IIT Bombay, for constructive criticism on the manuscript and interesting discussions.
Fig. 4. Thermal disinfection, lysis and helicase dependent amplification of M. tuberculosis (H37Rv) achieved in a single heat incubation step at 65 °C for 60 min. Lane 1: 10–300 bp DNA ladder. Lane 2: HDA reaction without any template DNA (negative control). Some non-specific primer-dimer formation can be seen though. Lane 3: HDA reaction in the absence of the amplification enzymes (negative control). Lanes 4 and 5: HDA of M. tuberculosis using purified genomic DNA as the starting material (positive controls). The DNA in lane 4 was extracted by mechanical lysis, whereas the DNA in lane 5 was obtained by chemical lysis. Lane 6: HDA using mechanically lysed M. tuberculosis (using FastPrep) cells as the starting material. HDA was performed on the lysate without DNA purification. Lanes 7–8: Results of two step thermal lysis and HDA of M. tuberculosis. Lanes 9–10: Results of one-step disinfection, thermal lysis and HDA of cultured M. tuberculosis. Lanes 7–10 shows results obtained using M. tuberculosis cells directly as starting material.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2017.09.013. 4
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