Journal Pone 0253235

PLOS ONE
RESEARCH ARTICLE
Rapid detection of multidrug-resistant

tuberculosis based on allele-specific
recombinase polymerase amplification and
colorimetric detection
Nuntita Singpanomchai1, Yukihiro Akeda2, Kazunori Tomono2, Aki Tamaru3,
Pitak Santanirand4, Panan Ratthawongjirakul ID5*
1 Program of Molecular Sciences in Medical Microbiology and Immunology, Department of Transfusion

a1111111111
Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok,
a1111111111 Thailand, 2 Division of Infection Control and Prevention, Osaka University Hospital, Osaka University, Osaka,
a1111111111 Japan, 3 Department of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan,
a1111111111 4 Microbiology Unit, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand,
a1111111111 5 Research Unit of Innovative Diagnosis of Antimicrobial Resistance, Department of Transfusion Medicine
and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand
* panan_etc@yahoo.com
OPEN ACCESS
Citation: Singpanomchai N, Akeda Y, Tomono K,

Abstract
Tamaru A, Santanirand P, Ratthawongjirakul P
(2021) Rapid detection of multidrug-resistant
Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to TB control. Early diag-
tuberculosis based on allele-specific recombinase nosis and proper treatment are essential factors to limit the spread of the disease. The exist-
polymerase amplification and colorimetric ing molecular tests for MDR-TB usually require specific instruments, steady power supply,
detection. PLoS ONE 16(6): e0253235. https://doi.
and routine maintenance, which might be obstacles for low-resource settings. This study
org/10.1371/journal.pone.0253235
aimed to develop allele-specific isothermal recombinase polymerase amplification (allele-
Editor: Seyed Ehtesham Hasnain, Indian Institute
specific RPA) to simultaneously detect the most common mutations in the rpoB gene at
of Technology Delhi, INDIA
codons 516, 526, and 531, which are associated with rifampicin resistance, and in the katG
Received: February 27, 2021
gene at codon 315, which is related to isoniazid resistance. Allele-specific primers targeting
Accepted: June 1, 2021 four major mutations, rpoB516, rpoB526, rpoB531, and katG315, were constructed and
Published: June 11, 2021 used in individual RPA reactions. The RPA amplicons were endpoints detected by the
Copyright: © 2021 Singpanomchai et al. This is an
naked eye immediately after applying SYBR Green I. The optimised RPA assay was evalu-
open access article distributed under the terms of ated with the Mycobacterium tuberculosis wild-type strain H37Rv and 141 clinical M. tuber-
the Creative Commons Attribution License, which culosis isolates. The results revealed that allele-specific RPA combined with SYBR Green I
permits unrestricted use, distribution, and
detection (AS-RPA/SYBR) detected these four major mutations with 100% sensitivity and
reproduction in any medium, provided the original
author and source are credited. specificity relative to DNA sequencing. The limits of detection for these particular mutations
with AS-RPA/SYBR were 5 ng. As a result of the outstanding performance of AS-RPA/
Data Availability Statement: All relevant data are
within the paper, its Supporting Information files, SYBR, including its easy setup, speed, lack of a specific instrument requirement, and lack of
and at NCBI Genbank. The accession numbers are cross-reaction with other bacteria, this technique may be integrated for the molecular diag-
1. accession number MZ208045-MZ208144 is for nosis of MDR-TB, especially in low-resource settings.
the rpoB gene 2. accession number MZ208146-
MZ208245 is for the katG gene.
Funding: This study was partially supported by the

scholarship from the Overseas Research
Experience Scholarship for Graduate Student and
the Ratchadaphiseksomphot Endowment Fund
PLOS ONE | https://doi.org/10.1371/journal.pone.0253235 June 11, 2021 1 / 15

PLOS ONE Rapid detection of MDR-TB based on allele-specific RPA
2016 of Chulalongkorn University (NRU-59-012- Introduction

HR). The funders did not play any role in the study
design, data collection and analysis, decision to Tuberculosis (TB) remains a significant health problem worldwide. In 2019, the World Health
publish, or preparation of the manuscript. Organization (WHO) estimated that approximately 10 million people developed TB disease
Competing interests: The authors have declared
and that approximately 1.4 million affected people died [1]. Multidrug-resistant tuberculosis
that no competing interests exist. (MDR-TB), defined as Mycobacterium tuberculosis that resists at least two of the most potent
medications against TB infection, rifampicin and isoniazid, is a challenge for TB control. In
2019, the incidence of multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB)
was estimated to be approximately 465,000 cases, while 206,030 cases were noted with a 57%
success rate for treatment outcomes [1]. MDR-TB expands the disease transmission period,
leading to increased morbidity, mortality, and treatment failure rates. Over 95% of rifampicin-
resistant isolates possess single point mutation clustering in the 81-base-pair hotspot region of
the rpoB gene known as a rifampicin-resistant determining region (RRDR) that encodes
codons 507 to 533. The highest global prevalence of mutations in the RRDR is at codons 531,
526, and 516 [2–4]. In contrast, mutations in several genes can lead to isoniazid resistance.
However, the majority (60–80%) of isoniazid resistance in M. tuberculosis is due to a single
point mutation in the katG gene at codon 315 (50–93% of katG mutations) [2, 5].
The crucial steps to limit TB spread are early detection and appropriate treatment [6, 7].
Molecular diagnostic approaches have been developed for rapid TB diagnosis and drug suscep-
tibility testing. However, several molecular methods mainly rely on amplification-based tech-
nologies such as PCR, which are being implemented in developing countries to shorten the
turnaround time of conventional phenotypic methods [8, 9]. Xpert MTB/RIF (Cepheid, Sun-
nyvale, CA, USA) has been introduced to developing countries for the simultaneous detection
of TB and rifampicin resistance through cartridge-based fully automated real-time PCR [10].
Although Xpert MTB/RIF is rapid and endorsed by the WHO for initial testing of MDR-TB
detection, some limitations, including a reliance on a stable power supply and regular mainte-
nance, impede its potential for application as a point-of-care test (POCT). Furthermore, high-
cost laboratory instrumentation and cartridges impact sustainability in the long term in coun-
tries with a high TB burden, including Thailand. Other molecular techniques for MDR-TB
detection, such as line probe assays, are PCR-based endpoint detection systems [11]. After
amplification, the amplicons are detected by test strips, which are lined with a broad set of
mutation-specific detection probes that complicate result presentation and hinder their practi-
cal use in routine diagnostics. Hence, rapid, easy-setup and low-cost MDR-TB screening sys-
tems used as POCTs in low-resource settings are urgently required.
Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplifica-
tion technique. With the benefit of two core enzymes (a recombinase and a strand displacing
DNA polymerase) and a single-stranded DNA-binding (SSB) protein, RPA does not depend
on the thermal denaturation of a template and operates at a low and constant temperature
[12]. Thus, thermocycler-independent amplification can be performed using only a low-cost
heating box, which is advantageous for application in low-resource settings. RPA has been
combined with either real-time- or endpoint-based detection for rapid and visual detection of
results, which was recently demonstrated for a potential portable POCT for TB and MDR-TB
[13–16].
This study’s main objective was to develop an easy-setup, rapid and affordable screening
diagnostic method for MDR-TB. Our method was based on allele-specific RPA and SYBR
Green I (SYBR), referred to as AS-RPA/SYBR. RPA was used in an isothermal amplification
process with a set of allele-specific primers with intentional mismatches that could discrimi-
nate hotspot single point mutations conferring rifampicin and isoniazid resistance. The targets
we specified were rpoB516, rpoB526, and rpoB531 for rifampicin resistance and katG315 for

isoniazid resistance. Our method avoided the requirement of electric-based detection; thus,
SYBR Green I was used to interpret amplified RPA products by observing a colour change by
the naked eye under visible light. The overall time to detection, i.e., the amplification process
to the read-out step, was only 30–40 minutes.
Materials and methods

Ethical approval
In this study, M. tuberculosis genomic DNA was extracted from discarded colonies after a rou-
tine diagnostic examination. All of the DNA samples were then fully anonymised with no
patient data links to protect patient confidentially before accessed by the researchers and used
in the subsequent processes. The study protocol was fully approved by the Research Ethics
Review Committee for Research Involving Human Research Subjects, Health Science Group,
Chulalongkorn University (Certificate of approval number 036/2018).
DNA controls
Genomic DNA of the standard M. tuberculosis reference strain H37Rv (ATCC25618) was used
as a wild-type control for the study. Genomic DNA obtained from a clinical M. tuberculosis
strain with known mutations at codons 516, 526, and 531 of the rpoB gene and codon 315 of
the katG gene (confirmed by Sanger DNA sequencing) was used as a mutant control. All geno-
mic DNA was kindly provided by the Microbiology Unit, Faculty of Medicine Ramathibodi
Hospital, Mahidol University, Bangkok, Thailand. The control DNA concentrations were
measured using a spectrophotometer (NanoDrop 8000, Thermo Scientific, USA). The control
DNA was stored at -20˚C until use.
DNA samples
A total of 141 genomic DNA samples extracted from leftover M. tuberculosis colonies were
used in this study. All M. tuberculosis was isolated from patients (one isolate/patient) from the
Microbiology Unit, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok,
Thailand (designated Thailand strain, n = 100) and the Department of Bacteriology, Osaka
Prefectural Institute of Public Health, Osaka, Japan (designated Japan strains, n = 41). All M.
tuberculosis samples were initially cultured by the MGITTM liquid culture system (Becton
Dickinson and Company, Franklin Lakes, NJ, USA) and differentiated between M. tuberculosis
complex and nontuberculous mycobacteria by the SD Bioline TB Ag MPT64 assay (Standard
Diagnostics, Gyeonggi-do, Republic of Korea). A phenotypic susceptibility test for rifampicin
and isoniazid was performed by the MGITTM liquid culture system.
DNA sequencing analysis

To analyse mutations associated with rifampicin and isoniazid resistance in all 141 M. tubercu-
losis DNA samples, two specific targets, the rpoB gene (nucleotide positions 1131–1570) and
katG gene (nucleotide positions 631–1260), were amplified by conventional PCR. Each reac-
tion contained 1X PCR Buffer, 0.2 μM each forward and reverse primer (Table 1), 200 μM
dNTPs, 1.5 mM MgCl2 and 1.5 U of Taq DNA Polymerase (New England Biolabs, Ipswich,
MA, USA). Amplification of both targets was performed with the following conditions: initial
denaturation at 95˚C for 5 minutes, 35 cycles of amplification (95˚C for 1 minute, 55˚C for
rpoB or 63˚C for katG for 1 minute, and 72˚C for 1 minute), and a final extension at 72˚C for 7
minutes. M. tuberculosis H37Rv genomic DNA and sterile distilled water were used as positive
and negative controls, respectively. The PCR products of both genes were analysed by 1.5%

Table 1. Primer sequences for PCR and DNA sequencing.

Genetic regions Primers Sequences (5’!3’) Product sizes (bp) References
rpoB rpoB F GCTGATCCAAAACCAGATCC 440 This study
rpoB R ACACGATCTCGTCGCTAACC
katG katG F AGCGGTAAGCGGGATCTGGAGAA 630 [17]
katG R CATGTCTCGGTGGATCAGCTTGTA
https://doi.org/10.1371/journal.pone.0253235.t001
agarose gel electrophoresis with UltraPower DNA/RNA Safedye (Gellex, Tokyo, Japan) and
visualised under an ultraviolet (UV) transilluminator. All the PCR products were sent to Bio-
neer Sequencing Service Co., Ltd., Republic of Korea, to perform sequence analysis by the con-
ventional Sanger method using an ABI 3730XL DNA Analyser (Applied Biosystems, Foster
City, CA, USA). Nucleotide sequences of the rpoB and katG genes from each of the 141 sam-
ples were aligned with the corresponding reference sequence of M. tuberculosis H37Rv (Gen-
Bank accession No. NC_000962) using BioEdit software version 7.2.6.
RPA primer design and screening

An RPA assay was developed targeting the most common rifampicin- and isoniazid-associated
mutations, which are located at codons 516, 526, and 531 of rpoB and codon 315 of katG,
respectively. To identify TB, the IS1081-F and IS1081-R primers were used to amplify an inser-
tion sequence, IS1081, that is highly specific for M. tuberculosis. For the rpoB gene, rpoB-F and
rpoB-R were designed to amplify the rpoB gene as a control. A set of rpoB allele-specific prim-
ers was designed for specific point mutation detection at codons 516, 526, and 531, which con-
fer rifampicin resistance. These primers included rpoB516-F and rpoB526-F (used in
combination with rpoB-R) and rpoB531-F (used in combination with rpoB-R2). For the katG
gene, katG-F and katG-R were designed to amplify the katG gene as a control. The KatG315-F
allele-specific primer (used in combination with katG-R) was used for specific point mutation
detection at codon 315, which was associated with isoniazid resistance.
RPA primers for the rpoB and katG gene sets were designed from the reference DNA
sequence of M. tuberculosis H37Rv (GenBank accession No. NC_000962) using the Primer3 pro-
gram (version 0.4.0; http://bioinfo.ut.ee/primer3-0.4.0/) with a modification for allele-specific
primers according to the Yaku-Bonczyk method [18]. The 3’ terminus of each forward allele-spe-
cific primer was specific to the wild-type alleles responsible for conferring drug resistance. If the
targeted location was wild-type, the allele-specific fragment was amplified. In contrast, if the tar-
geted location contained the mutation, amplification of the allele-specific fragment would be pre-
vented. An additional single-base mismatch at the third base from the 3’ terminus of each
forward allele-specific primer was added to improve the discriminatory power between wild-
type and mutant (S1 Appendix). These RPA primer candidates contained 29–33 bases and had
GC contents ranging from 56% to 62%, as recommended by the RPA manufacturer (TwistDx
Limited, Maidenhead, UK). The OligoAnalyser 3.1 program (https://eu.idtdna.com/calc/
analyser) and BLAST software (https://blast.ncbi.nlm.nih.gov/Blast.cgi) were used to assess the
possibility of secondary structure formation and the specificity of the primers, respectively.
Two to four forward and reverse primers for each target were designed in this study. Each
forward and reverse primer candidate was paired and preliminarily screened for its ability to
amplify specific targets with a TwistAmp1 Basic Kit (TwistDx Limited, Maidenhead, UK) as
recommended by the manufacturer. Once the most suitable primers were identified, they were
finally used in our developed RPA assay. Table 2 summarises all the final selected RPA primers
used in this study.

Table 2. RPA primer sequences and optimum conditions used in this study.
Genetic Primer Sequence (5’!3’) Product size Reference Final DNA Final primer concentration
region (bp) concentration (ng) (μmol/L)
IS1081 IS1081 F CCTCTTCTCATCTTATCGACGCCGAGCAGC 173 [16] 0.1 0.48
IS1081 R CTGATTGGACCGCTCATCGCTGCGTTCGC
rpoB rpoB F TCGGCGAGCTGATCCAAAACCAGATCCGGGTCG 363 This 0.1 0.4
rpoB516 F TCGGCACCAGCCAGCTGAGCCAATTCATCGA 213 study 0.1 0.5
rpoB526 F CCAGAACAACCCGCTGTCGGGGTTGACTCA 182 0.1 0.5
rpoB R CCGACAGCGAGCCGATCAGACCGATGTTGGGC
rpoB531 F TCGGGGTTGACCCACAAGCGCCGACTCTC 250 0.2 0.5
rpoB R2 ACACGATCTCGTCGCTAACCACGCCGTCG
katG katG F CTGATCGTCGGCGGTCACACTTTCGGTAAGACCC 276 This 0.1 0.2
katG315 F CCGGAACCGGTAAGGACGCGATCACCGG 152 study 0.1 0.5
katG R CTTGGCGGTGTATTGCCAAGCGCCAGCAGGGC
AS-RPA/SYBR optimisation
To determine the most appropriate conditions for RPA amplification and SYBR Green I detec-
tion, the optimised conditions were validated using genomic DNA of M. tuberculosis H37Rv (as
a wild-type control) and M. tuberculosis strains with known mutations within the rpoB and
katG genes (as mutant controls). During RPA assay development, the amount of DNA template
(0.00005–0.2 ng), primer concentration (0.2–0.6 μM pM), MgOAc concentration (14–20 mM),
incubation temperature (37–42˚C) and incubation period (15–40 minutes) were optimised
until the most appropriate conditions were obtained. SYBR Green I (250-1000X in a final vol-
ume of 25 μL of RPA products) was also validated to obtain the appropriate concentration. In
every optimisation, the RPA amplicons were examined by SYBR Green I and compared with
agarose gel electrophoresis. When observed with SYBR Green I, the condition giving clear read-
out results without nonspecific bands upon agarose gel electrophoresis was chosen.
AS-RPA/SYBR assay to detect rpoB and katG mutations

All 141 M. tuberculosis DNA samples were amplified by the RPA assay using a TwistAmp1
basic kit as recommended by the manufacturer. Genomic DNA of M. tuberculosis H37Rv and
M. tuberculosis strains with known mutations within the rpoB and katG genes were used as
wild-type and mutant controls, respectively. Amplification of the targets IS1081, rpoB,
rpoB516, rpoB526, rpoB531, katG, and katG315 was performed separately. Each RPA reaction
was composed of 29.5 μL of RPA rehydration buffer, 14 mM MgOAc, forward and reverse
primers (Table 2), DNA template, and sterile distilled water up to 50 μL. The RPA reactions
were incubated at 37˚C for 15 minutes. At the end of the amplification, RPA amplicons were
equally divided into two tubes, 25 μL each, for different endpoint detection methods.
The naked-eye detection of RPA amplicons was performed by immediately observing col-
orimetric changes in natural light after adding 1 μL of 375x SYBR Green I (TaKaRa Bio,
Tokyo, Japan) to 25 μL of RPA amplicons. In the presence of RPA amplicons, the colour of the
solution changed from the original orange to bright green, indicating that M. tuberculosis
DNA was detected or that no single point mutation occurred at the specific codons (indicating
that the isolate was probably a susceptible wild-type strain). On the other hand, the solution
remained orange in the absence of the RPA amplicon, implying that M. tuberculosis DNA was
not detected or that a single point mutation may have occurred at specific codons (indicating
that the isolate was probably a resistant mutant strain). The interpretation of RPA

amplification coupled with SYBR Green I detection was conducted independently by two dif-
ferent investigators who were blind to the results of the other test to prevent bias by the two
different investigators. For a precise comparison of the results, another 25 μL of the RPA
amplicons was purified by FavorPrep Gel/PCR Purification (Favorgen Biotech Corp., Ping-
Tung, Taiwan). The purified RPA amplicons were electroporated by 1.5% agarose gel electro-
phoresis with UltraPower DNA/RNA Safedye (Gellex, Tokyo, Japan) and visualised under UV
light. All experiments were performed in triplicate.
Limit of Detection (LOD)

The lowest DNA concentration that was detected by AS-RPA/SYBR was assessed. Genomic
DNA of M. tuberculosis H37Rv (ATCC25618) and clinical M. tuberculosis strains with known
mutations within the rpoB and katG genes (obtained from the Microbiology Unit, Faculty of
Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, with no patient data
links) was serially diluted tenfold from 5 to 0.00005 ng. Each DNA dilution was used as a tem-
plate for each primer set for RPA amplification. Sterile distilled water was used as a negative
control. After amplification, each RPA product was detected by both SYBR Green I and 1.5%
agarose gel electrophoresis.
Specificity testing
To analyse the specificities of AS-RPA/SYBR, genomic DNA extracted from pathogens that
commonly cause respiratory tract infection and other common Mycobacterium spp. was used
as a template. A total of 5 ng of DNA from the individual pathogen was used for each primer
set for RPA amplification. These pathogens included clinical Acinetobacter baumannii, Hae-
mophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa,
Streptococcus pneumoniae, Streptococcus pyogenes, Mycobacterium avium, and Mycobacterium
intracellulare strains. All of the above pathogens were kindly provided with no patient data
links by the Microbiology Unit, Faculty of Medicine Ramathibodi Hospital, Mahidol Univer-
sity, Bangkok, Thailand, and confirmed for species using biochemical testing and MALDI--
TOF MS, except for M. avium and M. intracellulare that confirmed by a 16S rRNA
sequencing. M. tuberculosis H37Rv (ATCC25618) genomic DNA and sterile distilled water
were used as positive and negative controls, respectively.
Results
Distribution of drug-resistant phenotypes among M. tuberculosis samples
Results of the phenotypic susceptibility tests of 141 M. tuberculosis isolates were provided by
the Microbiology Unit, Faculty of Medicine Ramathibodi Hospital, Bangkok, Thailand, and
the Department of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan.
These isolates included 73 strains that were rifampicin and isoniazid susceptible, 4 strains that
were rifampicin monoresistant, 14 strains that were isoniazid monoresistant, 31 strains that
were MDR-TB, and 19 strains that were extremely drug-resistant tuberculosis (XDR-TB)
(Table 3).
Sequencing analysis
Mutations conferring rifampicin and isoniazid resistance were explored by conventional Sanger
DNA sequencing of the rpoB and katG genes, respectively. Almost all rifampicin- and isoniazid-
susceptible strains did not possess mutations either in the rpoB or katG genes, except for one
strain that was rifampicin susceptible and had a mutation at rpoB codon 533 and one strain that

Table 3. Phenotypic drug susceptibility testing.

Strains Susceptible Rifampicin monoresistant Isoniazid monoresistant MDR-TB XDR-TB Total
Thai 73 4 14 9 0 100
Japanese 0 0 0 22 19 41
Total 73 4 14 31 19 141
was isoniazid susceptible and had a mutation at katG codon 315. Forty-six of 54 (83.19%)
strains resistant to rifampicin (rifampicin monoresistant, MDR-TB or XDR-TB) possessed
nucleotide mutations within the rpoB gene at the top three highest prevalence codons, codons
531 (28 strains, 51.85%), 516 (10 strains, 18.52%), and 526 (8 strains, 14.81%) (Table 4). Addi-
tional uncommon mutations within the rpoB gene included those at codon 511 (1 strain,
1.85%), codon 513 (2 strains, 3.70%), codon 522 (1 strain, 1.85%), and codon 533 (3 strains,
5.56%). Two rifampicin-resistant strains had no mutations observed within the 81-base-pair
hotspot region of the rpoB gene. Mutations of katG conferring isoniazid resistance (isoniazid
monoresistant, MDR-TB or XDR-TB) were found in 38 (59.38%) strains (Table 5), all of which
possessed amino acid substitutions at codon 315 (38 strains, 100%). Interestingly, 26 (40.62%)
isoniazid-resistant strains did not have a mutation within the katG gene.
Establishment of AS-RPA/SYBR assay

After various optimisations, the most appropriate condition for the AS-RPA/SYBR assay
was established. The assay included eight individual RPA reaction tubes. The optimised
assay showed that AS-RPA/SYBR was able to correctly amplify specific targets and effi-
ciently differentiate the wild-type and mutant templates. When validated with genomic
DNA of M. tuberculosis H37Rv (Fig 1), SYBR Green I remained orange in a negative control
tube due to no template DNA for amplification. In contrast, SYBR Green I turned bright
green in the remaining tubes, indicating that DNA products were amplified by RPA with
Table 4. Frequency of amino acid substitutions within 81-base-pair hotspot region of the rpoB gene of 141 M. tuberculosis.
rpoB gene
codons Amino acid substitutions Frequencies
Susceptible to rifampicin Resistance to rifampicin
No mutation 86 2
511 Leucine ! Proline 0 1
513 Glutamine ! Proline 0 2
516 Aspartic acid ! Valine 0 7
Aspartic acid ! Tyrosine 0 1
Aspartic acid ! Glycine 0 1
522 Serine ! Glutamine 0 1
526 Histidine ! Tyrosine 0 2
Histidine ! Aspartic acid 0 3
Histidine ! Arginine 0 2
Histidine ! Serine 0 1
531 Serine ! Leucine 0 28
533 Leucine ! Proline 1 2
516 and Aspartic acid ! Valine 0 1
533 Leucine ! Proline
Total (n = 141) 87 54

Table 5. Frequency of amino acid substitutions within the katG gene of 141 M. tuberculosis.
katG gene
codons Amino acid substitutions Frequencies
Susceptible to isoniazid Resistance to isoniazid
No mutation 76 26
315 Serine ! Threonine 1 37
Serine ! Asparagine 0 1
Total (n = 141) 77 64
specific primer sets. These test results indicate the detection of rifampicin- and isoniazid-
susceptible TB. However, when validated with M. tuberculosis strains with known mutations
within the rpoB and katG genes (Fig 2), SYBR Green I remained orange in a negative control
tube and rpoB531 and katG315 reaction tubes due to the targeted location containing the
mutation that inhibited amplification. SYBR Green I turned bright green in the rest of the
tubes. These test results indicate the detection of rifampicin- and isoniazid-resistant TB (or
MDR-TB). The presence of unaccounted faint smear bands on an agarose gel in Fig 2A
(lanes N, 5, and 7) did not lead to a false positive or background on SYBR Green I detection
and did not interfere with the assay.
Detection of rpoB and katG mutations using the AS-RPA/SYBR assay

The AS-RPA/SYBR assay was evaluated using 141 M. tuberculosis DNA samples. The
AS-RPA/SYBR assay results showed that 140 M. tuberculosis strains were positive with the
Fig 1. Results of the AS-RPA/SYBR assay validated with the M. tuberculosis wild-type strain. (A) RPA amplicons
were detected via agarose gel electrophoresis. The product sizes from each pair of primers were 173 bp, 363 bp, 213 bp,
182 bp, 250 bp, 276 bp and 152 bp for the IS1081, rpoB, rpoB516, rpoB526, rpoB531, katG and katG315 primers,
respectively. Lane L: 100-bp DNA ladder, Lane N: no template control; Lane 1: IS1081 primer; Lane 2: rpoB primer;
Lane 3: rpoB516 primer; Lane 4: rpoB526 primer; Lane 5: rpoB531 primer; Lane 6: katG primer; Lane 7: katG315
primer. (B) The naked-eye endpoint detection method was performed by adding SYBR Green I directly to the reaction
tubes. Tube N is a no-template control; tubes 1 to 7 contained the IS1081, rpoB, rpoB516, rpoB526, rpoB531, katG and
katG315 primers, respectively, showing a green colour, which implied a positive amplification result and no mutation
at those allele-specific sites.
https://doi.org/10.1371/journal.pone.0253235.g001

Fig 2. Results of the AS-RPA/SYBR assay when validated with M. tuberculosis strains with known mutations
within the rpoB and katG genes. This figure represents the results after testing with M. tuberculosis DNA containing
the rpoB531 and katG315 mutations. (A) RPA amplicons from different primers were detected via agarose gel
electrophoresis. The product sizes from each pair of primers were 173 bp, 363 bp, 213 bp, 182 bp and 276 bp for the
IS1081, rpoB, rpoB516, rpoB526 and katG primers, respectively. Reactions with the rpoB531 and katG315 primers
showed no visible target band. Lane N: no-template control; Lane 1: IS1081 primer; Lane 2: rpoB primer; Lane 3:
rpoB516 primer; Lane 4: rpoB526 primer; Lane 5: rpoB531 primer; Lane 6: katG primer; Lane 7: katG315 primer; Lane
L: 100-bp DNA ladder. (B) The naked-eye endpoint detection method was performed by adding SYBR Green I directly
to the reaction tubes. Tube N was a no-template control; tubes 5 and 7 were tested for rpoB531 and katG315,
respectively, showing an orange colour, which implied a negative amplification result. For allele-specific primers, these
results also indicated that the DNA template carried mutations at a particular site. Tubes 1–4 and 6 contained the
IS1081, rpoB, rpoB516, rpoB526 and katG primers, respectively, showing a green colour, which implied a positive
amplification result and no mutation at those allele-specific sites.
IS1081 primers, indicating TB detection, while only one strain was undetectable. However, the
rpoB and katG primer sets enabled the detection of all 141 M. tuberculosis strains. For the
allele-specific primer sets, 10, 8, 28, and 39 strains were negative with the rpoB516, rpoB526,
rpoB531, and katG315 primer sets, indicating that a single point mutation occurred at the spe-
cific codons (rifampicin and/or isoniazid resistance was detected).
Table 6. Validation of AS-RPA/SYBR by a DNA sequencing analysis (N = 141).

AS-RPA/SYBR assay DNA sequencing Sensitivity Specificity
Mutation detected Mutation not detected
rpoB516
Mutation detected 10 0 100% (95%CI: 69.15%-100%) 100% (95%CI: 97.22%-100%)
Mutation not detected 0 131
rpoB526
Mutation detected 8 0 100% (95%CI: 63.06%-100%) 100%(95%CI: 97.26%-100%)
rpoB531
katG315

Table 7. Validation of AS-RPA/SYBR by a phenotypic drug susceptibility test (N = 141).

AS-RPA/SYBR assay Rifampicin or Isoniazid susceptibility Sensitivity Specificity
tests
Resistant Susceptible
Either rpoB516 or rpoB526 or rpoB531
Mutation detected 46 0 85.19% (95%CI: 72.88%-93.38%) 100% (95%CI: 95.85%-100%)
katG315
Mutation detected 38 1 59.38% (95%CI: 46.37%-71.49%) 98.70% (95%CI: 92.98%-99.97%)
Finally, the AS-RPA/SYBR results were validated by DNA sequencing and a phenotypic
drug susceptibility test. When compared with the sequencing results (Table 6), both the sensi-
tivity and specificity of AS-RPA/SYBR assay were 100% for detecting codon 516 (95% CI:
69.15–100% and 95% CI: 97.22–100%), codon 526 (95% CI: 63.06–100% and 95% CI: 97.26–
100%) or codon 531 (95% CI: 87.66–100% and 95% CI: 96.79–100%) mutations of rpoB or
codon 315 (95% CI: 90.97–100% and 95% CI: 96.45–100%) mutation of katG.
However, when compared with the phenotypic drug susceptibility test results (Table 7), the
sensitivity and specificity of the AS-RPA/SYBR assay were 85.19% (95% CI: 72.88–93.38%)
and 100% (95% CI: 95.85–100%), respectively, for rifampicin resistance detection. However,
the sensitivity and specificity of the AS-RPA/SYBR assay were 59.38% (95% CI: 46.37–71.49%)
and 98.70% (95% CI: 92.98–99.97%), respectively, for isoniazid resistance detection.
LOD and specificity of the AS-RPA/SYBR assay

The LOD of each primer set in the AS-RPA/SYBR assay was 5 ng, except for the IS1081 prim-
ers, for which the LOD was 0.05 ng. The AS-RPA/SYBR assay did not cross-react with A. bau-
mannii, H. influenzae, K. pneumoniae, M. catarrhalis, P. aeruginosa, S. pneumonia, S. pyogenes,
M. avium, or M. intracellulare (Fig 3).
Discussion
One primary TB control strategy is early detection and prompt treatment [19]. However, the
emergence of MDR-TB is one of the factors that disrupts successful TB control. Rapid and
accurate MDR-TB diagnosis could guide appropriate treatment, decreasing morbidity and
mortality rates. Hence, we developed a novel, rapid, and easy-setup RPA assay to detect
MDR-TB, which can be easily used for routine diagnosis by low-resource-setting laboratories.
Our AS-RPA/SYBR assay targeted four major mutations, rpoB516, rpoB526, rpoB531, and
katG315, associated with rifampicin- and isoniazid-resistant TB.
Codons 531, 526, and 516 are the most common mutation sites among rifampicin-resistant
TB reported globally and confer a high level of rifampicin resistance (32 to 256 μg/mL). Other
mutations, such as at codons 522 and 533, have been occasionally reported, but they are associ-
ated with low-level resistance [20]. A previous study from Thailand showed that all 143
MDR-TB isolates had mutations at rpoB, with 98% of strains mutated in the hot-spot RRDR
and the remaining isolates mutated in the area of the N-terminus. Mutations at codons 531,
526, and 516 were found the most, with 58%, 25%, and 9% detection, respectively. Codon 526
exhibited the most variable nucleotide substitutions [3]. Isoniazid resistance is mainly associ-
ated with mutations in katG codon 315 in Thailand and other countries [2, 5]. Our results
showed a concordant result with an earlier study. However, the DNA samples tested in this

Fig 3. Specificity RPA testing. (A) Agarose gel electrophoresis showed the expected rpoB526 RPA product when
testing with wild-type M. tuberculosis H37Rv DNA only (lane 10). Lane L is a 100-bp DNA ladder, lane N is a no-
template control, and lanes 1–9 are DNA extracted from A. baumannii, H. influenzae, K. pneumoniae, M. catarrhalis,
M. intracellulare, S. pyogenes, S. pneumoniae, M. avium, and P. aeruginosa, respectively. (B) RpoB526 RPA products
were observed for the SYBR Green I colour change by the naked eye. Tube N is a no-template control, and tubes 1–9
are DNA extracted from A. baumannii, H. influenzae, K. pneumoniae, M. catarrhalis, M. intracellulare, S. pyogenes, S.
pneumoniae, M. avium, and P. aeruginosa, respectively, showing an orange colour, which indicates no amplification.
Tube 10 is M. tuberculosis H37Rv DNA, showing a green colour, which implies a positive amplification result.
study were derived from M. tuberculosis colonies isolated from leftover routine specimens.
They were not representative of the population attending the hospitals.
Loop-mediated amplification (LAMP) is an isothermal amplification recommended by the
WHO for the detection of TB. The LAMP test takes less than 1 hour, and its results can be read
by the naked eye under UV light [21]. Previous studies demonstrated that the application of
LAMP coupled with Au nanoprobes could detect specific mutation sites related to rifampicin and
isoniazid resistance in M. tuberculosis [22, 23]. RPA is another isothermal amplification that has
excellent sensitivity and specificity compared with that of conventional PCR. To the best of our
knowledge, there is no evidence comparing RPA to LAMP to directly detect M. tuberculosis from
specimens. However, RPA offers many advantages over other isothermal amplification tech-
niques, such as simple primer design, a short reaction time, a broad temperature range (25–
45˚C), and multiplexing availability [24]. With a minimal instrumentation requirement and a
wide range of storage temperatures (<-15˚C, 2–8˚C or 22–28˚C) and cold chain-independent
transportation, RPA is very suitable for basic laboratory settings or as a POCT [13, 25–29].
Rifampicin- and isoniazid-resistant TB occur mainly due to single point mutations in rpoB
and katG, respectively. Therefore, single-nucleotide polymorphism (SNP)-based detection has
been implemented in many novel generations of TB diagnostic tools. One simple system is
based on allele-specific amplification, which dramatically shortens SNP detection times com-
pared to those of sequencing-based platforms. Allele-specific primers have been used in the
RPA reaction to differentiate between wild-type and mutant [15, 27, 30]. Our established RPA
applied allele-specific primers combined with SYBR Green I to conduct instrument-free
nucleic acid amplification and MDR-TB detection. Other types of mutations such as deletion
and insertion can also be detected by allele-specific amplification through intentional multiple
base-pair mismatches between primer-template [31]. However, among the different types of
mutations, substitution rather than indels is the most significant mutation associated with
rifampicin- and isoniazid-resistant TB. Thus, allele-specific primer with additional single-base
mismatch exhibits sufficient discriminatory power to differentiate between wild-type and
mutant in our recent study.

AS-RPA/SYBR showed a high degree of specificity for MDR-TB diagnosis compared with
that for DNA sequencing. All four mutated gene targets corresponding to rifampicin and iso-
niazid resistance were identified with 100% specificity. The discriminatory power between the
wild-type and mutant was increased by adding a single mismatch of three base pairs from the
3’ terminus of the allele-specific primers [18]. Here, each forward allele-specific primer’s fur-
thest 3’ base was designed to specify the wild-type allele expected to anneal. The second base
was always annealed to either the wild-type or mutant allele. Furthermore, the third base was
an intentional mismatch that will never anneal to either type of allele. Previous studies showed
a consistent result between allele-specific RPA and DNA sequencing methods when detecting
a single-base mutation [27, 30]. When compared to the phenotypic drug susceptibility test,
AS-RPA/SYBR showed 59.38% katG315 detection. A katG315 mutation was identified in one
isoniazid-susceptible strain. However, this mutation was concordant with a result obtained
from DNA sequencing.
The sensitivity of the AS-RPA/SYBR assay reported here is high (100% sensitivity) com-
pared to that of DNA sequencing. Compared to a phenotypic drug susceptibility test, sensitiv-
ity decreased to 85.19% (for rifampicin) and 59.38% (for isoniazid). Our AS-RPA/SYBR assay
focused on detecting mutations only at codons 516, 526, and 531 within the RRDR of the rpoB
gene and codon 315 of the katG gene. Therefore, this assay cannot identify mutations outside
of these four target sites. Over 95% of rifampicin-resistant M. tuberculosis strains have a muta-
tion in the RRDR, and the most prevalent mutations (over 80%) affect codons 531, 526, and
516. Less than 5% of resistant strains do not show a mutation in the RRDR or other regions of
the rpoB gene [20]. Phenotypic isoniazid resistance has been related to multiple genes but
mostly katG (60–80%). A previous study from Thailand revealed mutations in katG, inhA, the
oxyR-ahpC intergenic region, and ndh in 80.6%, 13.8%, 2.5%, and 0.6% of cases, respectively.
Almost 98% of the katG mutations were serine substitution to threonine at codon 315. In addi-
tion, one interesting study revealed a higher percentage of Ser315Thr in MDR strains than in
isoniazid monoresistant strains [32]. Our AS-RPA/SYBR assay showed the potential to render
a rapid and specific method for detecting rifampicin resistance at rpoB gene positions 531, 526,
and 516 and isoniazid resistance at katG gene position 315, which covers the majority of muta-
tions associated with MDR-TB. Any amino acid substitution at these four target sites would
not affect the detection efficiency. Other mutated gene targets should be incorporated in the
assay to improve the sensitivity in the coverage of additional mutant strains. Besides, a sample
size of the most common codon, especially codon 516 and 526, should be expanded to improve
the confidence interval of the results.
AS-RPA/SYBR showed no cross-reaction with other bacterial pathogens. The LOD was 5
ng in a 50 μL RPA reaction. A higher sensitivity of 0.05 ng was reported in an RPA coupled
with SYBR-based detection of M. tuberculosis using an IS1081-specific primer in our previous
study [16]. The lower sensitivity observed in this study can probably be attributed to the nature
of the targeted gene detected. Allele-specific RPA may require template concentrations greater
than gene detection for successfully changed SYBR Green I colour observations [27]. Further-
more, the amplicon length might affect the LOD of SYBR Green I. The optimal DNA length
recommended for SYBR Green I detection is generally less than 200 bp [33].
RPA has been employed in many studies to detect the M. tuberculosis genome [13, 16, 34,
35]. However, to the best of our knowledge, only two publications demonstrate MDR-TB
detection by RPA integrated by lab-on-a-disc and a fluorescent detector [14, 15]. SYBR Green
I was chosen as the detection platform in our recent study to obtain instrument-free nucleic
acid amplification and detection for MDR-TB diagnosis. SYBR Green I is a cyanine dye used
as a nucleic acid stain for a detection platform in several studies. It preferentially binds to dou-
ble-stranded DNA, resulting in a DNA-dye complex that emits green light and can be detected

by the naked eye [33]. When SYBR Green I was conjoined with our RPA technique, this plat-
form’s outcomes can be achieved within only 30–40 minutes (from sampling to read out) with-
out any specific instrument requirements, which might be suitable for a POCT. In our
previous pilot studies, AS-RPA/SYBR was preliminarily validated with a patient sputum sam-
ple. The results showed that AS-RPA/SYBR required a high concentration and quality of DNA
template. Thus, a proper but straightforward extraction method for a patient sputum sample
should be developed. Moreover, AS-RPA/SYBR should be further validated with nucleic acid
from the direct specimens to fulfil the demand for a truly simple and rapid diagnosis of
MDR-TB.
Conclusion
Our study demonstrates that AS-RPA/SYBR clearly identified major specific point mutations
at codons 531, 526, and 516 of rpoB and codon 315 of katG, which are associated with rifampi-
cin and isoniazid resistance of M. tuberculosis, respectively. AS-RPA/SYBR is rapid and easy to
perform. Due to the lack of specific and expensive laboratory instrument requirements,
AS-RPA/SYBR can be implemented as a new molecular diagnostic method for detecting
MDR-TB. The application of AS-RPA/SYBR could limit the spread of MDR-TB, especially in
low-resource settings. A larger number of clinical samples should be evaluated to confirm the
test’s diagnostic performance and reliably apply it to routine laboratory applications.
Supporting information
S1 Appendix. How to design allele-specific RPA primer.
(DOCX)
S1 Raw images.
(PDF)
Acknowledgments
The authors would like to acknowledge the staff from the Clinical Microbiology Laboratory,
Faculty of Medicine Ramathibodi Hospital, Mahidol University, Thailand and the Department
of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan for providing M.
tuberculosis H37Rv and clinical strains DNA.
Author Contributions
Conceptualization: Panan Ratthawongjirakul.
Formal analysis: Nuntita Singpanomchai.
Funding acquisition: Panan Ratthawongjirakul.
Investigation: Nuntita Singpanomchai.
Methodology: Nuntita Singpanomchai.
Resources: Aki Tamaru, Pitak Santanirand.
Supervision: Yukihiro Akeda, Kazunori Tomono, Pitak Santanirand.
Writing – original draft: Nuntita Singpanomchai.
Writing – review & editing: Panan Ratthawongjirakul.

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PLOS ONE

Rapid detection of multidrug-resistant

1 Program of Molecular Sciences in Medical Microbiology and Immunology, Department of Transfusion

Citation: Singpanomchai N, Akeda Y, Tomono K,

Funding: This study was partially supported by the

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2016 of Chulalongkorn University (NRU-59-012- Introduction

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Materials and methods

DNA sequencing analysis

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Table 1. Primer sequences for PCR and DNA sequencing.

RPA primer design and screening

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AS-RPA/SYBR assay to detect rpoB and katG mutations

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Limit of Detection (LOD)

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Table 3. Phenotypic drug susceptibility testing.

Establishment of AS-RPA/SYBR assay

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Detection of rpoB and katG mutations using the AS-RPA/SYBR assay

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Table 6. Validation of AS-RPA/SYBR by a DNA sequencing analysis (N = 141).

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Table 7. Validation of AS-RPA/SYBR by a phenotypic drug susceptibility test (N = 141).

LOD and specificity of the AS-RPA/SYBR assay

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