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Rapid isothermal point mutation detection – towards a first pass screening strategy for multidrug resistant tuberculosis Benjamin Yong Chou Ng, Eugene J.H. Wee, Kyra Woods, Will Anderson, Fiach Antaw, Hennes Z.H. Tsang, Nicholas P. West, and Matt Trau Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01685 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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Analytical Chemistry

Rapid isothermal point mutation detection – towards a first pass screening strategy for multidrug resistant tuberculosis Benjamin Y.C. Ng,1,2 Eugene J.H. Wee,1 Kyra Woods,2 Will Anderson,1 Fiach Antaw,1 Hennes Z.H. Tsang,1 Nicholas P. West2* and Matt Trau1,2* 1

Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and

Nanotechnology, The University of Queensland, QLD 4072, Australia. 2

School of Chemistry and Molecular Biosciences, The University of Queensland, QLD

4072, Australia. *[email protected], [email protected]

ABSTRACT: Point mutations in DNA are useful biomarkers that can provide critical classification of disease for accurate diagnosis and to inform clinical decisions. Conventional approaches to detect point mutations are usually based on technologies such as real time PCR or DNA sequencing, which are typically slow and require expensive lab-based equipment. While rapid isothermal strategies such as recombinase polymerase amplification (RPA) have been proposed, they tend to suffer from poor specificity in discriminating point mutations. Herein, we describe a novel strategy that enabled exquisite point mutation discrimination with isothermal DNA amplification, using mismatched primers in conjunction with a two round enrichment process. As a proof-of-concept, the method was applied to the rapid and specific identification of drug resistant Mycobacterium tuberculosis using RPA under specific conditions. The assay requires just picogram levels of genomic DNA input, is sensitive and specific enough to detect 10% point mutation loading and can discriminate between closely related mutant variants within 30 minutes. The assay was subsequently adapted onto a low-cost 3Dprinted isothermal device with real time analysis capabilities to demonstrate a potential point-of-care application. Finally, the generic applicability of the strategy was shown by detecting three other clinically important cancer-associated point mutations. We believe that our assay shows potential in a broad range of healthcare screening processes for

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detecting and categorizing disease phenotypes at the point-of-care, thus reducing unnecessary therapy and cost in these contexts.

INTRODUCTION Point mutations in DNA are useful biomarkers that can provide critical information about the nature of the disease and inform clinical decisions. For instance point mutations can be used to identify drug resistant pathogens1 or subtype cancers2 which in turn, enable clinicians and healthcare workers to take appropriate actions. Conventional approaches to detect point mutations are usually based on technologies that possess exquisite sensitivity to DNA mismatches, such as real time PCR3 or DNA sequencing.4 However, such methods are typically slow and require expensive lab-based equipment, thus rendering them unsuitable for point-of-care diagnostic screening applications,5,6 where there is an increasing need for quick turnaround results to facilitate urgent healthcare processes.7 Isothermal DNA amplification technologies that run at a constant temperature, such as recombinase polymerase amplification (RPA)8, have recently garnered interest due to their excellent compatibility with point-of-care (POC) assays.9-11 RPA boasts similar sensitivity and specificity to conventional PCR. However, to date, RPA has been suggested as poorly suited for discriminating point mutations. Recent studies have reported that RPA is able to amplify targets with up to 9 primer-template mismatches,12 prompting suggestions for a highly polymorphic primer design to circumvent this limitation of low RPA specificity.13 Herein, we describe a novel nested RPA strategy to rapidly detect point mutations with exquisite discrimination. In order to improve the utility of the assay for on-site screening applications, our strategy avoids the use of expensive lab based devices such as thermal cyclers typical of conventional PCR-based methods. As a proof of concept, the nested RPA-based assay was used to detect and identify drug resistant (DR) Mycobacterium tuberculosis (Mtb) mutants – a significant and increasing problem in TB endemic regions.14 Multidrug resistant Mtb (MDR-TB) is defined as Mtb being resistant to both rifampicin (RIF) and isoniazid (INH), the two frontline drugs used to treat TB. In 2014, there were ~500,000 new cases of MDR-TB with an approximate 40% mortality rate.15 MDR-TB requires a much longer course of expensive and toxic drugs to treat.16 Currently, the gold standard for MDR-TB diagnosis is a 3 week long drug susceptibility testing with bacterial cultures. The Xpert MTB/RIF real time PCR platform (Cepheid),17


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which was endorsed by the WHO for MDR-TB diagnosis, has cost and complexity issues that limit its use in resource poor settings.5,18 Thus, an affordable and rapid screening diagnostic for TB and antimicrobial resistance could be useful for MDR TB management at POC.19,20 In this study, we report that our assay could potentially form part of the solution for a low-cost DR-TB screening system. The assay has the potential of detecting picogram levels of starting DNA sample input, ideal for a first pass TB screening assay, with TB positive results being followed up with a second RPA reaction to discriminate point mutations associated with drug resistance with high accuracy. We also performed our screening assay on a low cost isothermal heating device to demonstrate its potential for POC. While we also believe that our assay is amenable to DNA extraction techniques suitable for a POC strategy, further development along the lines of sample processing may be warranted. In addition, we demonstrated a generic approach for RPA-based point mutation assays by detecting various clinically important melanoma mutations.21-23 We believe that the nested RPA approach to improving discrimination of point mutations could have wide applications in both research and the clinic.

EXPERIMENTAL SECTION Materials. TwistAmp Basic RPA Kit was purchased from TwistDX (Cat# TABAS03KIT). All other chemicals were purchased from Sigma-Aldrich. DNA sample acquisition. M. tuberculosis H37Rv and both Leucine (Leu) and Tyrosine (Tyr) mutants of the β subunit of bacterial RNA polymerase (rpoB) were cultured in Middlebrook 7H9 complete broth with supplementation (10% ADC, 0.2% glycerol and 0.02% tyloxapol). Samples for assay testing were taken at mid-exponential phase (OD600 = 0.8). These samples were serially diluted and plated on Middlebrook 7H11 agar (0.5% glycerol and 10% OADC) and the number of colony forming units (CFU) was visually determined. All oligonucleotides, including gene blocks of the Leu and Tyr mutants and primers were purchased from Integrated DNA Technologies (IDT) and sequences are provided in Table S1. DNA extraction and purification. Genomic DNA of M. tuberculosis H37Rv, Leu and Tyr mutants were extracted directly from culture. Briefly, 1.5 mL of culture at mid log phase was centrifuged and the supernatant discarded. Pellet was resuspended in 700 µL of TE buffer,



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and bead beating was performed on the Beadbug homogenizer (Benchmark Scientific) for 90 seconds for mechanical lysis of Mtb cells. 500 µL of bead beat supernatant was transferred to a new tube and phenol-chloroform extraction of DNA was performed and repeated 3 times, before being treated with RNAse A. The extracted DNA was then ethanol precipitated and resuspended in 10mM Tris pH 8.0. Primary amplification of nucleic acid target. Nucleic acid amplification was performed with the TwistAmp Basic RPA Kit (TwistDX) as recommended by the manufacturer with some modifications. 12.5 µL reactions were performed at 37°C for 15 min using 1 µL of the nucleic acid extraction, 800 nM of each primer (outer primers) and 280 mM magnesium acetate. Following amplification, 3 µL of the RPA reaction was visualized by standard gel electrophoresis. Second round amplification of nucleic acid target. Nucleic acid amplification was performed as in the primary amplification, with modifications. Each of the 12.5 µL reactions was performed at 37°C for 15 min using 1 µL aliquot of primary amplification reaction, 400 nM of forward primer (standard for both mutant strains), 800 nM of reverse primer (mismatched to wild type), 2 nM of SYTO9 intercalating dye (Life Technologies) and 350 mM magnesium acetate. qRPA was performed on a Life Technologies 7500 qPCR machine set to a constant 37°C for 15 min with fluorescence acquired every 30 s. RESULTS AND DISCUSSION Enabling specific point mutation detection by RPA: Through a serendipitous experiment, we observed that RPA could specifically discriminate between two synthetic DNA sequences that differed by a single base under specific conditions. The first condition was that mutant-specific primers required a discriminating base at the 3’-end and an artificial mismatch of the third nucleotide from the 3’-end. While this primer design was a common strategy for allele-specific PCR,24 recent evidence in the literature suggests

that

RPA

still

occurs

indiscriminately

with

mismatches

at

these

positions.12,13,25,26 The second condition was that specific discrimination occurred only with enrichment of the target sequence, which could be a key prerequisite to enable rapid, highly specific point mutation discrimination by RPA. In practice, sequence enrichment is achieved by performing a “nested RPA” where outer primer pairs amplify the region of interest while inner primers interrogate the mutation of interest.


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To test the hypothesis, we compared the discriminatory ability of RPA with and without enrichment by performing the reaction with our mismatched primers on equimolar of target mutant synthetic DNA and non-target mutant synthetic DNA, with wild type genomic DNA as positive control (Fig. S1). As expected, specific discrimination was only possible with nested RPA where fonly the target mutant amplified. This thus confirmed our hypothesis that the enrichment of target sequence was a key prerequisite for highly specific point mutation discrimination by RPA, and fits into the assay design for a first round quick screening test for all types of TB (Fig. 1).

Figure 1. (A) Schematic representation of the nested RPA strategy. An outer amplification enriches the region of interest, which then allows for accurate point mutation discrimination in the inner amplification through the use of a primer with two mismatches, an artificial one at the third position from the 3’ end and a mutant mismatch at the 3’ end. (B) Diagnostic workflow.

The discriminatory effect of a nested RPA could be further enhanced by screening for most destabilizing combinations of 3’end mismatches (Fig. S2), adjusting primer concentrations (Fig. S3), and varying magnesium concentrations (Fig. S4).

Detecting drug resistance (DR) in TB with nested RPA: Compatibility of RPA for POC TB screening has been demonstrated in various studies.9-11 However, RPA has not been



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applied to DR-TB screening and could be a candidate proof-of-concept application for nested RPA. To this end, a nested RPA approach was conceived (Fig. 1). The purpose of a primary amplification is two-fold. Firstly, as the outer region was designed to lie outside all known mutations causing drug resistance, it functioned as a rapid screening test for TB independent of DR status. This provided timely and critical information for an appropriate response involving quarantine. Second, the primary amplification enriched for the target gene region that in turn facilitates the specific discrimination between positive (mutant) and negative (wild type) samples in a second round of RPA. In our study, we have selected the two most common point mutations in the β subunit of RNA polymerase (rpoB) associated with rifampicin resistance, namely, the C→T point mutation at codon 526 (Tyr mutant) and the C→T point mutation at codon 531 (Leu mutant).1,27 Together, these mutations account for greater than 75% of rifampicin resistance cases.1 We elected to use dsDNA-specific fluorescent dye as it provided cheap, rapid and convenient quantitation of RPA-generated DNA.28 This was in contrast to newer technologies such as labelled probes commonly used in electrochemical or SERS detection that drive up the cost and complexity of the assay. Moreover, this enables the adaptation of the assay onto our low-cost heating device, thus showing promise of a complete assay delivered at the POC.

Stringency of Point Mutation Detection in Heteroresistant Samples: In order to determine the primary amplification sensitivity of our assay in detecting TB, we performed standard RPA on a dilution series of Mtb mutant DNA with our outer primer set. We observed that we were able to detect as little as 1000 copies of Mtb mutant DNA, or 1 fg of DNA according to estimates that one genomic equivalent (GE) of Mtb is 5 fg of DNA (Fig. 2A). We further characterized the performance of our assay in detecting DR variants in a heteroresistant sample. We performed quantitative RPA on heteroresistant Mtb samples of proportions ranging from 0.1% to 100% mutant DNA, by spiking in known quantities of mutant DNA template to wild type Mtb DNA in order to simulate an enriched biologically complex sample. We were able to detect as low as 10% (1000 copies) mutant

Mtb DNA in a Mtb DNA complex based on comparison between the fluorescence onset times between the various heteroresistant samples (Fig. 2B). This was seen for both Tyr and Leu mutant-specific assays.


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Figure 2. Sensitivity of assay. (A) Limit of detection for primary amplification. Mutant Mtb gene was serial diluted 10-fold (L-R) number of copies: 108, 107, 106, 105, 104, 103, no template control and 103 of wild type genomic DNA (GE of 50 pg). (B) Limit of detection for heteroresistant samples of (top) Leu mutant and (bottom) Tyr mutant. Mutants are spiked in various concentrations (100%, 50%, 10%, 1%, 0.1%, 0% and no template control). Each figure is representative of at least 3 experimental replicates. (C) Primary amplification of (L-R) 104 copies of mutant DNA, 104 copies of WT genomic DNA (GE of 50 pg), combined template (10% heteroresistance), and no template control.

Based on the results, we spiked in 10% mutant gene (1000 copies) in 100% wild type Mtb genomic DNA (10000 copies or 50 pg), and performed our outer RPA reaction to generate our biologically complex sample (Fig. 2C) that was used for quantitative ∆t measurements.

Quantitative Analysis Using Internal Control: In a quantitative PCR (qPCR) reaction, an endogenously expressed housekeeping gene target was used as control for sample



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loading. This is also true for our assay particularly due to the inherent variation in the quality and quantity of Mtb DNA in a patient sample, which will affect the levels of fluorescence after the primary and second round amplification steps. To address this, we included a RPA assay targeting a highly conserved region in the amplicon enriched by the first RPA round. By measuring the difference in the time taken for fluorescence onset between the internal control and the mutant-specific assay (Δt), we were able to determine if the mutation was present. For our assays, we arbitrarily defined ∆t as the time it takes the sample to achieve the same level of fluorescence that the internal control achieved at 5 minutes. Based on our results across a range of concentrations, a ∆t ≤ 0 safely screens for mutant samples, and most heteroresistant samples (Fig. 3A, B). We found that most of the 100% mutant samples that we have tested produced negative ∆t values, indicating that they amplified faster than the internal controls. Samples that lag behind the internal control at 5 minutes can be safely regarded as a wild type strain or a rare event of heteroresistance. An additional benefit of the internal control RPA was that it also served to confirm the validity of the primary RPA for the presence of TB.

Figure 3. Determining ∆t with internal controls. To arrive on consensus for ∆t, assay was performed on three different concentrations (1, 10 and 100M) copies of Mtb DNA, with heteroresistance ranging from 100%, 50%, 10% and 0% MT (100% WT). Mutant samples for both (A) Leu and (B) Tyr achieved a ∆t < 0. Error bars represent


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standard error of at least 3 experimental replicates. (C) Primary amplification of genomic DNA extracted from wild type Mtb (W), Leu mutant (L), Tyr mutant (T) and no template control. (D) Second round amplification of WT and Leu mutant genomic DNA and (E) WT and Tyr mutant genomic DNA, both post- primary amplification. Both mutants achieved a ∆t < 0, in concordance with earlier results.

Discriminating drug resistant Mtb mutants: As a proof of concept, we performed our assay on drug resistant Mtb mutant strains. We first performed a primary amplification, to enrich for the region of interest, on genomic DNA extracted and purified from both Leu and Tyr mutants, and the wild type Mtb (Fig. 3C). A positive amplification also served to confirm that all 3 samples were TB positive. Our results were in concordance with the internal control tests, with all mutants having a ∆T ≤ 0, and wild type strain with a minimum ∆T of 1 minute. Notably, the difference in ∆T between the mutant and wild type Mtb genomic DNA were 3 minutes for the Leu mutant (Fig. 3D) and 2 minutes for the Tyr mutant (Fig. 3E). Since the doubling time of RPA was approximately 30 seconds,28 this is equivalent to a difference of around 4 and 6 cycles in a conventional qPCR system for the Leu and Tyr mutants respectively.

Low-cost Isothermal Device: To meet the needs of nucleic acid amplification tests at the POC, we built a prototype low-cost (~US$20) isothermal heating device that was able to maintain a temperature suitable for both rounds of amplification (37°C) (Fig. 4A). This device was equipped with an inexpensive LED and photo resistor combination to detect fluorescence levels. To test the limits of the machine and as a proof of concept, we performed our assay on a wild type and a Tyr mutant sample. The mutant achieved a ∆t = -30 s (Fig. 4B), while the wild type was ∆t = 1.5 min (Fig. 4C) and is consistent with our defined threshold for mutant calling on the commercial qPCR platform. These data suggested robust transferability of the assay across platforms and also the potential for low cost POC TB/MDR-TB screening.



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Figure 4. Proof of concept demonstrating assay applications. (A) Assay was performed on a low cost isothermal heating device with fluorescence readout. (B) 10% Tyr mutant (red) achieved target fluorescence in less than a minute of internal control (blue). (C) 100% WT template (red) achieved target fluorescence 1.5 minutes after internal control (blue). Green and purple lines represent no template controls for mutant and internal control primers respectively for both (A) and (B).

Application of Assay on Melanoma Subtypes: To demonstrate the broad applicability of the nested RPA strategy for point mutations in other diseases such as cancer, we designed assays against the clinically important BRAF V600E,23,29 NRAS Q61K21 and c-KIT L576P22,30 mutations. We found that all 3 mutations were easily identifiable from WT by gel electrophoresis (Fig. 5) thus demonstrating that the nested RPA approach was a generic approach to enabling rapid yet specific isothermal point mutation detection.


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Figure 5. Gel electrophoresis image of assay performed on three known point mutation targets for melanoma and their corresponding wild type templates. Lanes 13: BRAF V600E mutant, WT and no template control, Lanes 4-6: c-KIT L576P mutant, WT and no template control. Lanes 7-9: NRAS Q61K, WT and no template control.

CONCLUSION In conclusion, we have described a novel nested RPA strategy for rapid isothermal point mutation detection with exquisite specificity. This is in contrast to recent evidence in the literature that RPA is unsuitable for discriminating highly similar sequences. We believe that the proposed method couple to a cheap isothermal device could be a means to a low cost, rapid yet specific tool for point mutation diagnostics such as screening for TB/MDR-TB at POC.

Supporting Information. Primer design and selection, gel electrophoresis images for effects of primary amplification on specificity, effects of primer and magnesium acetate concentrations on stringency, brief description of isothermal heating device. (File name: Supporting Information revision). REFERENCES (1) Ramaswamy, S.; Musser, J. M. Tuber. Lung Dis. 1998, 79. (2) Pleasance, E. D.; Cheetham, R. K.; Stephens, P. J.; McBride, D. J.; Humphray, S. J.; Greenman, C. D.; Varela, I.; Lin, M. L.; Ordonez, G. R.; Bignell, G. R.; Ye, K.; Alipaz, J.; Bauer, M. J.; Beare, D.; Butler, A.; Carter, R. J.; Chen, L. N.; Cox, A. J.; Edkins, S.; KokkoGonzales, P. I.; Gormley, N. A.; Grocock, R. J.; Haudenschild, C. D.; Hims, M. M.; James, T.; Jia, M. M.; Kingsbury, Z.; Leroy, C.; Marshall, J.; Menzies, A.; Mudie, L. J.; Ning, Z. M.; Royce, T.; Schulz-Trieglaff, O. B.; Spiridou, A.; Stebbings, L. A.; Szajkowski, L.; Teague, J.; Williamson, D.; Chin, L.; Ross, M. T.; Campbell, P. J.; Bentley, D. R.; Futreal, P. A.; Stratton, M. R. Nature 2010, 463, 191-U173.



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(25) Abd El Wahed, A.; El-Deeb, A.; El-Tholoth, M.; Abd El Kader, H.; Ahmed, A.; Hassan, S.; Hoffmann, B.; Haas, B.; Shalaby, M. A.; Hufert, F. T.; Weidmann, M. PLoS One 2013, 8. (26) Abd El Wahed, A.; Patel, P.; Faye, O.; Thaloengsok, S.; Heidenreich, D.; Matangkasombut, P.; Manopwisedjaroen, K.; Sakuntabhai, A.; Sall, A. A.; Hufert, F. T.; Weidmann, M. PLoS One 2015, 10. (27) Telenti, A.; Imboden, P.; Marchesi, F.; Lowrie, D.; Cole, S.; Colston, M. J.; Matter, L.; Schopfer, K.; Bodmer, T. Lancet 1993, 341, 647-650. (28) Wee, E. J. H.; Trau, M. ACS Sens. 2016, 1, 670-675. (29) Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.; Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.; Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature 2002, 417, 949-954. (30) Beadling, C.; Jacobson-Dunlop, E.; Hodi, F. S.; Le, C.; Warrick, A.; Patterson, J.; Town, A.; Harlow, A.; Cruz, F.; Azar, S.; Rubin, B. P.; Muller, S.; West, R.; Heinrich, M. C.; Corless, C. L. Clin. Cancer. Res. 2008, 14, 6821-6828.

Author contributions B.N. planned and performed all experiments, interpreted results and wrote the manuscript. E.W. planned experiments, interpreted results and revised the manuscript. K.W. was instrumental in generating mutant TB cell lines. W.A. and F.A. conceived, programmed, and optimized the low cost isothermal device. H.T. contributed experimental results on mutations associated with melanoma subtypes. N.W. and M.T. were involved in giving advice over the course of the project and in performing the final revision of the manuscript.



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Isothermal Point Mutation Detection: Toward a First ... - ACS Publications

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