Proficient Detection of Multi-Drug-Resistant Mycobacterium

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Proficient detection of multidrug-resistant Mycobacterium tuberculosis by padlock probes and lateral flow nucleic acid biosensors Pavankumar R Asalapuram, Anna Engström, Jie Liu, David Herthnek, and Mats Nilsson Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04312 • Publication Date (Web): 17 Mar 2016 Downloaded from http://pubs.acs.org on March 18, 2016

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Analytical Chemistry

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Proficient detection of multidrug-resistant Mycobacterium tuberculosis by padlock

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probes and lateral flow nucleic acid biosensors

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Asalapuram R Pavankumar, Anna Engström§, Jie Liu#, David Herthnek, Mats Nilsson*

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Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm

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University, Stockholm, Sweden.

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*Corresponding author: Mats Nilsson, Science for Life Laboratory, Department of

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Biochemistry and Biophysics, Stockholm University, Box 1031, SE-17121 Stockholm,

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Sweden. E-mail: [email protected]; Phone: +46 (0)762 756 161.

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Present address:

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University, Uppsala, Sweden and Molecular and Experimental Mycobacteriology, Research

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Center Borstel, Borstel, Germany, #School of Chinese Medicine, Hong Kong Baptist

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University, China.

§

Department of Medical Biochemistry and Microbiology, Uppsala

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ACS Paragon Plus Environment

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Abstract

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Tuberculosis is a major communicable disease. Its causative agent, Mycobacterium

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tuberculosis, becomes resistant to antibiotics by acquisition of point mutations in the

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chromosome. Multidrug-resistant tuberculosis (MDR-TB) is an increasing public health

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threat and prompt detection of such strains is of critical importance. As rolling circle

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amplification of padlock probes can be used to robustly distinguish single-nucleotide

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variants, we combined this technique with a sensitive lateral flow nucleic acid biosensor to

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develop a rapid molecular diagnostic test for MDR-TB. A proof-of-concept test was

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established for detection of the most common mutations [rpoB 531 (TCG/TTG) and katG 315

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(AGC/ACC)] causing MDR-TB and verification of loss of the respective wild type. The

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molecular diagnostic test produces visual signals corresponding to the respective genotypes

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on lateral flow strips in approximately 75 min. By detecting only two mutations, the test can

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detect about 60% of all MDR-TB cases. The padlock probe-lateral flow (PLP-LF) test is the

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first of its kind and can ideally be performed at resource-limited clinical laboratories. Rapid

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information about the drug-susceptibility pattern can assist clinicians to choose suitable

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treatment regimens and take appropriate infection control actions rather than prescribing

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empirical treatment, thereby helping to control the spread of MDR-TB in the community.

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Introduction

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Tuberculosis (TB) is a major infectious disease causing 9 million new cases and 1.5 million

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deaths per year.1 The causative agent of TB, Mycobacterium tuberculosis (MTB), develops

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drug resistance by acquisition of mutations in the chromosome.2 MTB strains resistant to at

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least the two major first line anti-TB drugs isoniazid (INH) and rifampicin (RIF) are

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classified as multidrug resistant TB (MDR-TB). According to the 2014 WHO Global

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Tuberculosis Report, 3.5% of new and 20.5% of previously treated cases are infected with

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MDR-TB strains and the prevalence is expected to increase. MDR-TB treatment is very

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complicated and involves the use of more toxic drugs with prolonged treatment duration from

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months to years. Recently the TBNET and RESIST-TB networks suggested that the

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identification of MTB mutations in clinical isolates coding for katG, inhA, rpoB, embB, rrs,

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rpsL and gyrA has implications for the management of TB patients, pending the results of in

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vitro DST.3 Therefore, improved infection control measures and better diagnostic methods

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are needed to reduce the spread of MDR-TB strains. Correct identification of MDR-TB

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isolates remains a challenge for resource-limited clinical laboratories due to high cost of

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sample processing. Therefore, there is an urgent need of robust and inexpensive MDR-TB

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diagnostic test methods suitable for resource limited peripheral laboratories to facilitate

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initiation of early and effective treatment.

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Generally, MTB is primarily detected by smear microscopy, which is a relatively cheap and

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rapid method. However, it cannot identify drug-resistant strains. Culture-based methods for

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phenotypic drug susceptibly testing (DST), on the other hand, are laborious and time-

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consuming, requiring weeks to months to obtain results due to the slow growth of MTB.4 In

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contrast, polymerase chain reaction (PCR) based nucleic acid amplification tests (NAAT)

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provide rapid results for the detection of drug-resistant MTB strains through various


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automated and semi-automated techniques. For example, the GeneXpert MTB/RIF (Cepheid,

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Sunnyvale, CA, USA) provides rapid indicative identification of MDR-TB directly from

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sputum samples in less than 2 hours.5 However, it is restricted to only specific detection of

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RIF resistance. The probe-hybridization assay, GenoType MTBDRplus (Hain Lifescience

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GmbH, Nehren, Germany) can detect MDR-TB,6 but requires multiple manual steps and

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specific instruments. Although the solid-phase reverse hybridization line-probe assay (LPA)

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can rapidly detect RIF and INH resistance, it is not recommended by WHO for the detection

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of second-line drug-resistance attributes.7-8 Conclusively, the capacity and requirement for

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instrumentation and skilled personnel of currently available NAATs hinder prompt detection

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of MDR-TB in resource-limited settings. Therefore, a proficient method that can address the

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molecular detection challenges of MDR-TB and be implemented in peripheral laboratory

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settings is required to provide easily interpreted first-hand information about MDR-TB

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isolates.

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In the light of the need of rapid diagnostic methods, the use of lateral flow nucleic acid

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biosensors (LFNAB) becomes an excellent choice, on which immobilized antibodies or

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nucleic acid tags on a nitrocellulose membrane specifically bind to their corresponding

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antigens or nucleic acid targets. The visual signals (color change) are usually developed on

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the lateral flow (LF) strips by means of hybridization of substrates like streptavidin, biotin,

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horseradish peroxidase, or conjugated gold nanoparticles (AuNP).9-10 Generally, the target

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molecules are amplified by an isothermal NAAT,11 and in case of drug-resistant TB, the

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main challenge lies in the correct identification of point mutations. Among several potential

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isothermal amplification methods, rolling circle amplification (RCA) in combination with

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target recognition by padlock probes (PLPs) is one of the most promising technologies that

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enable accurate detection of single nucleotide variants, even in a highly multiplexed


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fashion.12 PLPs are linear oligonucleotide probes that enable mutation detection by ligation-

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dependent circularization.13-15 As the probe ends hybridize in juxtaposition on the target

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specific site, a perfect match at the 3’ end is required for ligation, efficiently discriminating

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point mutations.16 The linker segment in the middle contains sequences with functions for

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amplification, identification and detection. The circularized padlock can undergo RCA to

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produce a single-stranded concatemer, containing multiple complementary repeats of the PLP

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sequence. In order to improve the sensitivity, the concatemer is restriction digested, re-ligated

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into new circles and subjected to an additional round of RCA, known as circle-to-circle

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amplification (C2CA).17 Requirement of sophisticated instruments like fluorometers,

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fluorescence microscopes, array scanners, etc. to read out C2CA signals hamper the use of

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RCA in resource-limited laboratories and calls for alternative methods.

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Earlier, we have developed a molecular method for the multiplex detection of RIF-resistant

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MTB based on padlock probes and magnetic nanobeads.18 However, due to the singleplex

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nature of the applied readout, wild type confirmation and species identification had to be

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performed in different reaction tubes. Considering the advantages of RCA in molecular

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diagnostics, especially to discriminate the single nucleotide variants in drug-resistant MTB,

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we have previously combined the method with LFNAB to produce multiplex qualitative

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color signals for the identification of INH-resistance targeting katG 315 mutation. In this

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study, we present the proof-of-concept for a simple, specific and cost-effective diagnostic

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PLP-LF test for the prompt identification of MDR-TB from primary cultures. The DNA-

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based test was developed for the application in resource-limited clinical laboratories that

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gives a conception about the drug resistance pattern of both INH and RIF, which would be

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very valuable for clinicians in order to take appropriate actions for treatment and infection

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control.


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Experimental section

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Chemicals and oligonucleotides

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Streptavidin from Streptomyces avidinii, gold(III)chloride trihydrate (HAuCl4), sucrose,

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dithiothreitol (DTT), Triton X-100, trisodium citrate, Tris(hydroxymethyl)aminomethane

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hydrochloride (Tris-HCl), Tween 20, Ethylenediaminetetraacetic acid (EDTA) and bovine

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serum albumin (BSA, for oligonucleotide-AuNP conjugates) were purchased from Sigma-

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Aldrich (St. Louis, MO, USA). Sodium chloride-sodium citrate (SSC) buffer (pH 7.0),

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phosphate buffer saline (PBS, pH 7.4, 0.01 M), and sodium chloride (NaCl, 5 M, pH 7.0)

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were purchased from Substratenheten at Klinisk mikrobiologi, Karolinska University hospital

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(Stockholm, Sweden). ATP and dNTP were purchased from Thermo Scientific (Waltham,

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MA, USA). All oligonucleotides were purchased from Integrated DNA Technologies

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(Leuven, Belgium) and Sigma-Aldrich. For the LF assay, binder-free borosilicate glass fiber

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pads (grade A/C), cellulose fiber absorbent pads (grade 113) and nitrocellulose membrane

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(BioTrace™ NT) attached to the laminated cards/strips (0.4 and 0.5 cm width) were

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purchased from Kinbio Tech (PuDong, Shanghai, China).

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Bacterial strains and DNA extraction

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The reference strain MTB H37Rv (ATCC 25618) and ten clinical MTB isolates (Table 1),

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previously genotyped by Engström et al.,19 were cultured on Löwenstein-Jensen medium with

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and without 40 mg/L of RIF, respectively. DNA was extracted according to Juréen et al.,20

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and 10 µg of genomic DNA was fragmented enzymatically using 10 U each of NaeI and

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HpyCH4V, and 1x CutSmart buffer (New England Biolabs, Ipswich, MA, USA) at 37°C for

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90 min followed by enzyme inactivation at 65°C for 20 min. DNA concentration was

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measured by the dsDNA HS and BR assays using Qubit 2.0 fluorometer (Life Technologies,

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Carlsbad, CA, USA).


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Padlock probes and rolling circle amplification

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Sequences of oligonucleotides used in this study are given in Table 2. Four PLPs were

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designed to target two codons in the genes katG and rpoB and their corresponding wild type

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sequences (katG 315 ACC (MUT), katG 315 AGC (WT), rpoB 531 TTG (MUT) and rpoB

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531 TCG (WT). Secondary structure predictions were analyzed using Mfold Web Server.21

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The PLPs were phosphorylated at the 5’ end by incubating a reaction mixture consisting of 1

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µM oligonucleotide, 1x PNK buffer A, 1 mM ATP), and 1 U/µl T4 polynucleotide kinase

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(Thermo Scientific, Waltham, MA, USA) at 37°C for 30 min, followed by enzyme

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inactivation at 65°C for 20 min. Confirmation of PLP efficacy was done by performing

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C2CA, according to Dahl et al.,17 with minor modifications: the coiled amplification products

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produced from 1 amol of synthetic target was fluorescently labeled and quantified by

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automated counting in a microfluidic cell under a confocal microscope (data not shown).22

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Sensitivity of the assay was evaluated by LF strips with amplicons prepared from 300 pg, 3

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ng, 30 ng and 300 ng of genomic DNA. The specificity of the PLPs were confirmed by

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performing C2CA on 300 ng genomic DNA extracted from the strains in Table 1.

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As depicted in Figure 1A, the C2CA assay procedure starts by hybridization and ligation at

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60°C for 5 min in 20 µL of reaction mixture containing target DNA, 1x Ampligase buffer,

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250 mU/µL Ampligase (EpiCenter, Madison, WI, USA), 0.2 µg/µL BSA, 33 nM of each of

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the phosphorylated PLPs and 50 nM of each capture probe. Streptavidin-coated magnetic

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beads (Dynabeads MyOne Streptavidin T1, Life Technologies, California, USA) were

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washed three times in 1x Wtw buffer [10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% Tween

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20, 0.1 M NaCl] and ligated circles were coupled to 5 µl of these beads by means of the

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biotinylated capture probes at room temperature (RT) for 5 min. Unreacted oligonucleotides

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were removed by washing the beads once with 100 µl of 1x Wtw buffer and the buffer was


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replaced by 20 µL of RCA mixture [0.2 µg/µL BSA, 125 µM dNTP, 1x Phi29 buffer and 100

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mU/µL Phi29 DNA polymerase (Thermo Scientific, Waltham, MA, USA)]. RCA was

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performed at 37°C for 20 min followed by enzyme inactivation at 65°C for 1 min. The single

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stranded concatemers were monomerized by incubation in a digestion mixture consisting of

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120 nM of restriction oligonucleotide S00244, 0.2 µg/µL BSA and 120 µU/µL of AluI (New

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England Biolabs, Ipswich, MA, USA) and 1x Phi29 buffer followed by incubation at 37°C

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for 5 min and enzyme inactivation at 65°C for 3 min. The beads were discarded and

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monomerized RCA products were re-circularized and amplified again in 1x Phi29 buffer, 0.2

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µg/µL BSA, 100 µM dNTP, 0.68 mM ATP, 60 mU/µL Phi29 DNA polymerase and 14

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mU/µL T4 DNA ligase (Thermo Scientific, Waltham, MA, USA) at 37°C for 20 min,

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followed by enzyme inactivation at 65°C for 1 min. For application on LF strips, the C2CA

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concatemers were again monomerized by adding a digestion mixture [1.8 µM restriction

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oligonucleotide S00166, 0.2 µg/µL BSA and 550 µU/µL AluI in 1x Phi29 buffer] at 37°C for

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10 min, followed by enzymatic inactivation at 65°C for 3 min.

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Preparation and characterization of oligonucleotide conjugated gold nanoparticles

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Gold nanoparticles were prepared by a standard citrate reduction method with slight

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modifications.9,23 In a dry 500 mL round-bottom borosilicate glass flask, cleaned in

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aquaregia (nitric acid and hydrochloric acid in 3:1 ratio), 100 mL of 0.01% HAuCl4 in

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MilliQ water was boiled with vigorous stirring. Four milliliters of 1% trisodium citrate

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solution was added and after turning wine-red, the solution was boiled for 10 more min.

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Heating was turned off and the AuNP-solution was allowed to gradually cool to RT and was

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stored at 4°C, until further use.

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Fifty micromolar of thiolated oligonucleotide, designed to hybridize to a sequence present in

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all C2CA monomers, was reduced by 500 mM of DTT in SSC buffer for 30 min at RT. A

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NAP™-5 column (GE Healthcare Biosciences, Little Chalfont, UK) was equilibrated with

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citrate buffer and the activated oligonucleotide was eluted directly into 1 mL of 4-fold

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concentrated AuNPs. After gentle mixing, it was incubated for 2 h at 37°C and 50 µL of 1 M

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NaCl was added with gentle agitation. It was kept for ‘aging’ at 4°C for 24 h and another 100

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µL of 1 M NaCl was added. The solution was centrifuged at 13,000 g for 25 min, the

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supernatant discarded and the AuNP-oligonucleotide conjugates re-dispersed in 1 mL of 5%

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BSA, 0.25% Tween 20 and 20 mM Tris-HCl (pH 8.0) and filtered through a 0.2 µm syringe

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filter (Merck Millipore KGaA, Darmstadt, Germany).

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Conventional transmission electron microscope (TEM) images of the prepared AuNP were

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obtained at 100 kV with low-dose procedures and magnifications of 50,000x and 300,000x

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using JOEL JEM-2100 LaB6 microscope. The AuNP-oligonucleotide conjugates were

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characterized by measuring their light absorption at 520 nm in a Multi-Mode Microplate

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Reader (SpectraMax® M5, Molecular Devices). In addition, their size-distribution and

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surface charge (ζ-potential) was measured in citrate buffer (pH 7.0) at 25°C by dynamic light

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scattering (DLS) using Zetasizer Nano ZS90 (Malvern, UK) equipped with a 4.0 mW HeNe

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laser and an avalanche photodiode detector.

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Design, assembly and preparation of lateral flow strips

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As depicted in Fig. 1B, the 100 x 5 mm LF strip consists of a sample application pad,

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nitrocellulose membrane and absorbent pad that are mounted on a thin plastic backing. The

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dry sample pad (25 x 5 mm) was saturated using saturation buffer (1% BSA, 1% Triton

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X-100, 20 mM Tris-HCl, 100 mM NaCl; pH 8.0), air-dried and fixed on one end of the


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nitrocellulose membrane (45 x 5 mm) with an overlap of 2-3 mm and the absorbent pad (30 x

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5 mm) was fixed on the other end of the nitrocellulose membrane. The biotinylated strip

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oligonucleotides (Table 2) were immobilized in test and control zones on the nitrocellulose

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membrane. The control zone contained one line of immobilized oligonucleotides

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complementary to the AuNP-oligonucleotide conjugates. The test zone contained 4 lines

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separated by 3 mm, where each line consisted of unique strip oligonucleotides for detection

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of the C2CA monomers corresponding to their specific genotypes of katG 315 WT, katG 315

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MUT (AGC/ACC), rpoB 531 WT and rpoB 531 MUT (TCG/TTG) by hybridization (Fig.

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1B). Fifty micromolar of the strip oligonucleotide was mixed with an equal volume of 1x

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PBS containing 15 µM of streptavidin. After incubation at 37°C for 2 h, 3.9 nL of the

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streptavidin-conjugated oligonucleotide was printed (immobilized) on the nitrocellulose

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membrane using a nanoplotter (Nano-Plotter NP2.0, GeSiM, Grosserkmannsdorf, Germany).

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The strips were incubated overnight at 37°C and stored in a dry place until further use.

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Visualization of C2CA amplicons on lateral flow strips

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Fifty-five microliters of the C2CA monomers were hybridized with 13 µL of AuNP-

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oligonucleotide conjugates for 5 min at RT and applied to the sample pad of LF strips, drop

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by drop. The sample was allowed to flow for 5-7 min and washed with 4x SSC buffer for

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visualization of the red color bands. Color development in the control line indicated the

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positive assay control, while the signals from each test line specifically referred to presence

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of WT and/or MUT genotypes of katG 315 and rpoB 531. Intensity graphs were generated

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for the bands and pixel-densities were measured to quantify the results in densitograms, using

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the open source tool ImageJ (Version 1.49q).24

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Results and discussion

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There is an urgent need for development of prompt and inexpensive diagnostic tests for the

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correct identification of drug-resistant MTB strains. We aimed to develop a robust and

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stringent molecular diagnostic method for screening of MDR-TB, by combining PLP-RCA

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and LF biosensors. The test, as illustrated in the Fig. 1, is an assay format to produce rapid

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visual signals to discriminate between wild type and the most common mutations in katG and

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rpoB genes, causing MDR-TB, i.e. resistance to INH and RIF.

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Figure 1. Proof-of-principle of the PLP-LF assay

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The linker segment (grey) of the PLP contains detection and restriction sites, while the 5’ and

260

3’ arms (black) are designed to hybridize to the target sequence (orange; A). Upon matched


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hybridization, PLPs are ligated and the circularized probes can undergo RCA. The amplicons

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are digested using a restriction oligonucleotide. The products are again amplified by RCA

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(C2CA), monomerized and hybridized in a sandwich fashion to their common tags of AuNP

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oligonucleotides and to the respective WT or MUT oligonucleotide tags immobilized on the

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LF strips to produce visual signals (B). Blue are the restriction oligonucleotides, which will

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serve as templates to produce C2CA concatemers, which are further restriction digested to

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apply on lateral flow strips. Visual signals are developed when the immobilized streptavidin-

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bound oligonucleotides are hybridized to its respective C2CA monomers and thiolated

269

oligonucleotides conjugated with gold nanoparticles.

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The method can produce visual signals in approximately 75 min and the results can guide

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clinicians in taking informed decisions on public health control actions as well as adjusting to

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an effective antibiotic regimen. Hence, the test could be a preliminary alternative to the time-

274

consuming conventional DST and offers a compatible solution for resource-limited clinical

275

laboratories.

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Evaluation of AuNP-oligonucleotide conjugates

278

The TEM images of the AuNP (Fig. 2 A and B) confirm the size of sphered particles to be 15

279

± 3.5 nm. Size distribution curve based on the light absorption of AuNP showed a λ-max at

280

520 nm before oligonucleotide conjugation and the peak shifted to 527 nm after the

281

conjugation (Fig. 2C). However, the DLS measurements of AuNP-oligonucleotide conjugate

282

(Fig. 2D) revealed an average diameter of 90 ± 4 nm with a single peak indicating

283

monodispersed solution without particle aggregation. The ζ-potential measurements of -37.4

284

mV and -34.5 mV for AuNP and their oligonucleotide-conjugates, respectively, showed that

285

the preparations were stable.

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Figure 2. Analysis of gold nanoparticles and its oligonucleotide conjugates

291

Transmission electron microscopic (TEM) images at 50,000x and 300,000x magnification (A

292

and B) shows that the gold nanospheres have an average diameter of 15 ± 0.5 nm. A shift

293

light absorbance spectra (C) from 520 nm to 528 nm shows that the prepared gold particles

294

are conjugated with the oligonucleotides. Single smooth spectrum of DLS measurements (D)

295

confirms conjugation and monodispersion of the AuNP-oligonucleotide conjugates.

296 297

Limit of detection of padlock probe-lateral flow test

298

Limit of detection (LOD) of this PLP-LF assay was performed in triplicates by testing 10-

299

fold dilutions of extracted genomic DNA from the reference strain MTB H37Rv. Signal from

300

as little as 3 ng of DNA could be seen for the katG 315 WT probe, while at least 30 ng of


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DNA was needed for the rpoB 531 WT probe. Since both signals were clearly observed with

302

300 ng of DNA (Fig. 3A), further experiments with DNA from clinical samples (Table 1)

303

were performed using this amount. Densitograms (Fig. 3B) based on the local pixel density

304

of red color signals developed on lateral flow strips correlated with the visual observation.

305 306

Figure 3. LOD of PLP-LF method

307

Limit of detection of the PLP-LF method was investigated by testing 10-fold dilutions of

308

genomic MTB DNA (A). The densitograms in B, plotted by computing the local color

309

intensities, depicts the semi-quantitative changes in the control and test lines.

310 311

The signal intensity of rpoB 531 WT was lower compared to katG 315 WT, which could be

312

due to the high GC content in the target region of the rpoB gene, potentially resulting in a

313

lower yield of C2CA monomers. Minor variations were observed among the densitograms of

314

control lines, even though the lateral flow strips from the same printing session were used.

315

Such differences can be expected in LF tests25 due to the accumulation of AuNP around the

316

printed spots, differences in absorbance or flow properties of nitrocellulose membrane or the

317

position of printed lines. These problems will be addressed by improving the flow kinetics in

318

future work to increase the sensitivity of the test. Another approach to achieve higher

319

sensitivity would involve the addition of another cycle of RCA to the assay,26 which could


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enable direct testing on sputum samples.

321 322

Visual evaluation of the padlock probe-lateral flow test on clinical isolates of MTB

323

A set of 10 clinical isolates MTB (Table 1) that were resistant and/or susceptible to INH and

324

RIF, were tested by the PLP-LF method. As seen in Fig. 4, five strains (ID numbers: 2, 4, 8,

325

9, and 12) contained mutant katG and rpoB codons; three strains (19, 20 and 21) possessed

326

only wild type codons and one strain (13) showed the presence of wild type katG codon but

327

mutant rpoB codon.

328

329 330

Figure 4. Testing MTB clinical isolates by PLP-LF method

331

Ten characterized genomic DNA samples (300 ng) isolated from clinical MTB containing

332

wild type or mutant codons of rpoB 531 and katG 315 were tested. The densitograms

333

qualitatively shows the presence and/or absence of wild type and/or mutant genotypes of the

334

clinical isolates.

335 336

While sample number 17 clearly showed a line corresponding to mutant katG codon, it did

337

not yield a result for the rpoB codon on any replicate of the strips. This inconclusive result

338

demonstrates the usefulness of including probes targeting wild type in the test, since a lack of


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mutant rpoB 531 signals would otherwise have been interpreted as an indication of wild type

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rpoB. All the other visual signals of PLP-LF assay of the tested strains were fully concordant

341

to genotypic characterization of the strains by pyrosequencing19 and the respective

342

densitograms below each LF strip correspond to their visual signals. The method could be

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expanded by including a variety of other mutations causing MDR-TB, and the sensitivity

344

could be improved for application directly on sputum samples from TB patients.

345 346

Conclusion

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Drug-resistant TB is an increasing global health problem and the identification of MDR-TB

348

remains a challenge. Culture-based DST and advanced NAATs can only be performed in

349

well-equipped laboratories or require expensive equipment. In general, DST reports of anti-

350

TB drugs would miss important information about the non-concordant resistance-associated

351

mutations. As suggested in the TBNET/RESIST-TB consensus statement-2016, identification

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and provision of details about the MTB mutations causing drug-resistance in the genes like

353

katG, inhA, rpoB, embB, rrs, rpsL and gyrA would help the clinicians and health-care

354

professionals to take appropriate TB controlling measures. In order to establish such a robust

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diagnostic method offering reliable initial screening of MDR-TB that is suitable for resource-

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limited healthcare settings and peripheral clinical laboratories, we have developed a PLP-LF

357

assay. This unique and rapid test provides preliminary information about the most common

358

clinically significant mutations causing MDR-TB. Consequently, providing a valuable

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preview of the drug-susceptibility pattern at the early stages of diagnosis could certainly help

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clinicians to start treatment with appropriate antibiotics in as little as 75 min. In this proof-of-

361

concept study, we have included only the most prevalent mutations causing INH and RIF

362

resistance. This could be the first report of its kind to develop a visual detection test for the

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prompt identification of MDR-TB isolates, which can be potentially expanded for the


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detection extensively drug-resistant TB (XDR-TB) in resource-limited laboratories and

365

peripheral laboratories.

366 367

Acknowledgements

368

This research was partially supported by an Indo-Swedish cooperative program of Swedish

369

Research Council (VR), the Swedish Governmental Agency for Innovation Systems

370

(Vinnova) and Innovative Medicines Initiative, a public-private partnership between the

371

European Union, and the European Federation of Pharmaceutical Industries and Associations

372

(RAPP-ID project, grant agreement, no. 115153). Authors thank German Salazar Alvarez,

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(Institutionen för material- och miljökemi, Stockholm University) for helping with TEM

374

images, Teresa Zardań Gómez Torre (Department of Engineering Sciences, Nanotechnology

375

and Functional Materials, Uppsala University) for DLS measurements and Camilla Russell

376

(Dept. of Biochemistry and Biophysics, Stockholm University) for the useful technical

377

discussions and timely help.

378 379

Author Contributions

380

ARP, JL and MN conceived the concept; ARP, AE and LJ designed and performed the

381

experiments. ARP, AE, JL, DH and MN analyzed the data, and wrote the manuscript.

382 383

Conflict of interest

384

MN holds stock in Olink Bioscience AB that holds commercial rights to padlock probes.

385 386 387 388


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(1) Pai, M.; Schito, M.; J. Infect. Dis. 2015, 211(S2), S21–28. (2) Ramaswamy, S. L.; Musser, J. M. J. Tuber. Lung. Dis. 1998, 79, 3-29. (3) Domínguez, J.; Boettger, E. C.; Cirillo, D.; Cobelens, F.; Eisenach, K. D.; Gagneux, S.; Hillemann, D.; Horsburgh, R.; Molina-Moya, B.; Niemann, S.; Tortoli, E.; Whitelaw, A.; Lange, C.; for the TBNET and RESIST-TB networks. Int. J. Tuberc. Lung. Dis. 2016, 20, 24–42. (4) Kim, S. J. Eur. Respir. J. 2005, 25, 564–569. (5) Helb, D.; Jones, M.; Story, E.; Boehme, C.; Wallace, E.; Ho, K.; Kop, J. A.; Owens, M. R.; Rodgers, R.; Banada, P.; Safi, H.; Blakemore, R.; Lan, N. T. N.; 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. J. Clin. Microbiol. 2010, 48, 229-237. (6) Hillemann, D.; Rüsch-Gerdes, S.; Richter, E. J. Clin. Microbiol. 2007, 45, 2635-2640. (7) Wilson, M. L. Arch. Pathol. Lab. Med. 2013, 137, 812–819. (8) Theron, G.; Peter, J.; Richardson, M.; Barnard, M.; Donegan, S.; Warren, R.; Steingart, K. R.; Dheda, K. Cochrane. Database Syst. Rev. 2014, 10, CD010705. (9) Mao, X.; Ma, Y.; Zhang, A.; Zhang, L.; Zen, L.; Liu G. Anal. Chem. 2009, 81, 1660– 1668. (10) Mdluli, P.; Tetyana, P.; Sosibo, N.; Walt, H. V.; Mlambo, M.; Skepu, A.; Tshikhudo, R. Biosens. Bioelectron. 2014, 54, 1–6. (11) Deng, H.; Gao, Z. Anal. Chim. Acta 2015, 853, 30–45. (12) Nilsson, M.; Dahl, F.; Larsson, C.; Gullberg, M., Stenberg. J. Trends Biotechnol. 2006, 24, 83-88. (13) Nilsson, M.; Larsson, C.; Stenberg, J.; Göransson, J.; Grundberg, I.; Isaksson. M. Mol. Diagnostic. 2010, 117–132. (14) Nilsson, M.; Malmgren, H.; Samiotaki, M.; Kwiatkowski, M.; Chowdhary, B. P., Landegren, U. Science 1994, 265, 2085–2088. (15) Nilsson, M.; Krejci, K.; Koch, J.; Kwiatkowski, M.; Gustavsson, P.; Landegren, U. Nat. Genet. 1997, 16, 252–255. (16) Luo, J.; Bergstrom, D. E.; Barany, F. Nucleic Acids Res. 1996, 24, 3071–3078. (17) Dahl, F.; Baner, J.; Gullberg, M.; Mendel-Hartvig, M.; Landegren, U.; Nilsson, M. Proc. Natl. Acad. Sci. USA 2004, 101, 4548–4553. (18) Engström, A, Gómez de la Torre, Z. T.; Strømme, M.; Nilsson, M.; Herthnek, D.; PloS One 2004, 4, e62015. (19) Engström, A.; Morcillo, N.; Impriale, B.; Hoffner, S. E.; Jureen, P. J. Clin. Microbiol. 2012, 50, 2026-2033. (20) Jureen, P.; Engstrand, L.; Eriksson, S.; Alderborn, A.; Krabbe, M.; Hoffner, S. E. J. Clin. Microbiol. 2006, 44, 1925–1929. (21) Zuker, M. Nucleic Acids Res. 2003, 31, 3406–3415. (22) Jarvius, J.; Melin, J.; Go, J; Stenberg, J.; Fredriksson, S.; Gonzalez-Rey, C.; Bertilsson S. Nat. Methods 2006; 725–727. (23) Frens, G. Nat. Phys. Sci. 1973, 241, 20–22. (24) Schneider, C. A, Rasband, W. S.; Eliceiri, K. W. Nat. Methods. 2012, 9, 671-675. (25) Posthuma-Trumpie, G.; Korf, J.; Amerongen, A. V. Anal. Bioanal. Chem. 2009, 393, 569-582. (26) Ke, R.; Zorzet, A.; Göransson, J.; Lindegren, G.; Sharifi-Mood, B.; Chinikar, S.; Mardani, M.; Mirazimi, A.; Nilsson, M. J. Clin. Microbiol. 2001, 49, 4279–4285.


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Tables

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Table 1. Genotypic information of the clinical MTB strains used in the study Strain ID rpoB genotype katG genotype 2 S531L (TCG/TTG) S315T (AGC/ACC) 4 S531L (TCG/TTG) S315T (AGC/ACC) 8 S531L (TCG/TTG) S315T (AGC/ACC) 9 S531L (TCG/TTG) S315T (AGC/ACC) 12 S531L (TCG/TTG) S315T (AGC/ACC) 13 S531L (TCG/TTG) WT 17 S531L (TCG/TTG) S315T (AGC/ACC) 19 WT WT 20 WT WT 21 WT WT

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Table 2. Oligonucleotides used in this study Name

Sequence (5' --> 3')

Oligo ID S01233 S01234 S01237 S01239 S01235 S01236

rpoB 531 wt AtagrpoB 531 TTG AtagkatG 315 wt AtagkatG 315 ACC AtagTB-control oligo_v1 TB-Gold Oligo_v1

AAAAAAAAAAAAAAAGATCACACTTACGGAACAGC AAAAAAAAAAAAAAAGATCTAACGCACGGGAACTC AAAAAAAAAAAAAAACTGAGAGTTCGATGACCTGT AAAAAAAAAAAAAAAAATGCTCGGGAAGGCTACTC ATAGTGTCTTACTTAAAAAAAAAA GTAAGACACTATTACTGAGGAGAAAAAAAAAA

S00448

katG 315 wt

TGGTGATCGCGTCCTTACCACAGGTCATCGAACTCTCAGGTGTAT GCAGCTCCTCAGTAATAGTGTCTTACATACGACCTCGATGCCGC

S00565

katG 315 ACC

TGGTGATCGCGTCCTTACCGAGTAGCCTTCCCGAGCATTGTGTATG CAGCTCCTCAGTAATAGTGTCTTACATACGACCTCGATGCCGG

Padlock probe

S00592

rpoB 531 wt RS Popeye2

Padlock probe

S00228

rpoB 531 TTG RS v1

GGCGCTGGGGTTGCTGTTCCGTAAGTGTGATCGTGTATGCAGCTC CTCAGTAATAGTGTCTTACTGGTTGACCCACAAGTTTTTCCGACTG TC GGCGCTGGGGGAGTTCCCGTGCGTTAGATCGTGTATGCAGCTCCT CAGTAATAGTGTCTTACGCGCCGACTGTT

L11783

TB rpoB CO RS

L11860

katG CO

S00244 S00166 L12879

BNL_RO_AluI AluI RO TB rpoB SW RS wt v2

L11801

TB rpoB SW RS mut

L12721

katG SW wt v2

L12560

katG 315 ACC target

CTCTCTCTCTCTCTCTCTCTGTCCGCGACGTGCACCCGTCGCACTA CGGCCGGATGTGCC CTCTCTCTCTCTCTCTCTCTTTCCAGCCCAAGCCCATCTGCTCCAGC GGAGCAGCCTCGGGTTC GTGTATGCAGCTCCTCAGTA TACTGAGGAGCTGCATACAC GGCACATCCGGCCGTAGTGCGACGGGTGCACGTCGCGGACCCTCA CGTGACAGACCGCCGGGCCCCAGCGCCGACAGTCGGCGCTTGTGG GTCAACCCCGACAGCGG GGCACATCCGGCCGTAGTGCGACGGGTGCACGTCGCGGACCTCCA GCCCGGCACGCTCACGTGACAGACCGCCGGGCCCCAGCGCCAAC AGTCGGCGCTTGTGGGTCAACCCCGACAGCGGGTTGTT GAACCCGAGGCTGCTCCGCTGGAGCAGATGGGCTTGGGCTGGAA GAGCTCGTATGGTAAGGACGCGATCACCAGCGGCATCGAGGTCGT ATG GAACCCGAGGCTGCTCCGCTGGAGCAGATGGGCTTGGGCTGGAA GAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATCACCACCG GCATCGAGGTCGTATGGAC

Description Strip oligonucleotide Strip oligonucleotide Strip oligonucleotide Strip oligonucleotide Strip oligonucleotide AuNP-oligonucleotide conjugate Padlock probe

5' modification Biotin Biotin Biotin Biotin

3' modification

Biotin Thiol

Padlock probe Capture oligonucleotide

Biotin

Capture oligonucleotide

Biotin

Restriction oligonucleotide Restriction oligonucleotide Synthetic target Synthetic target Synthetic target Synthetic target

20 ACS Paragon Plus Environment

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TOC

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Proficient Detection of Multi-Drug-Resistant Mycobacterium

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