Research Article Development of Au-Nanoprobes Combined ...downloads.hindawi.com/journals/jchem/2016/3474396.pdf · Isoniazid Resistance in Mycobacterium tuberculosis JutturongCkumdee,

Research ArticleDevelopment of Au-Nanoprobes Combined withLoop-Mediated Isothermal Amplification for Detection ofIsoniazid Resistance in Mycobacterium tuberculosis

Jutturong Ckumdee1 Thongchai Kaewphinit2

Kosum Chansiri3 and Somchai Santiwatanakul4

1Department of Clinical Pathology Faculty of Medicine Vajira Hospital Navamindradhiraj University Samsen RdBangkok 10300 Thailand2Innovative Learning Center Srinakharinwirot University Sukhumvit 23 Bangkok 10110 Thailand3Department of Biochemistry Faculty of Medicine Srinakharinwirot University Sukhumvit 23 Bangkok 10110 Thailand4Department of Pathology Faculty of Medicine Srinakharinwirot University Sukhumvit 23 Bangkok 10110 Thailand

Correspondence should be addressed toThongchai Kaewphinit thongchaikaswuacthand Somchai Santiwatanakul titi41yahoocom

Received 8 March 2016 Revised 25 August 2016 Accepted 28 August 2016

Academic Editor Mostafa Khajeh

Copyright copy 2016 Jutturong Ckumdee et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Multidrug resistant tuberculosis (MDR-TB) isMycobacterium tuberculosis that does not respond to isoniazid and rifampicin so thecondition worsens continuously and creates difficulties for treatment by public health control programmes especially in developingcountries The real time polymerase chain reaction (PCR) combined with agarose gel electrophoresis or strip tests is usefulmolecular tools for diagnosis of MDR-TB Novel loop-mediated isothermal amplification (LAMP) can also detect drug resistancewhich is a one-point mutation by designing inner primers of 51015840 end specific with the mutant Au-nanoprobes on hybridisationwith LAMP products containing target-specific sequences remain red whereas test samples without specific sequences in the probeturn purple due to salt-induced aggregation of the Au-nanoprobes In this study a strategy was designed based on the LAMP of aDNA sample coupled to specific Au-nanoprobes which showed the potential to provide a rapid and sensitive method for detectingisoniazid resistance at katG gene position 315 (GrarrC) 46 clinical samples were tested and showed 100 specificity and sensitivitycompared with GenotypeMDR-TB PlusThis method was advantageous because it is rapid cheap specific and sensitive Furtherit does not require thermal cycles for MDR-TB detection

1 Introduction

Mycobacterium tuberculosis (MTB) infection has become anemerging global health concern According to the WorldHealth Organisation (WHO) Global Tuberculosis Report2012 there were an estimated 87 million cases of TB in 2011[1] Worldwide 37 of newly diagnosed incidences and 20of cases which have previously been treated are thought tobe a form of tuberculosis which has developed resistance tomultiple drugs (MDR-TB) defined as resistance to at leastisoniazid (INH) and rifampicin (RMP) In 2011 there werean estimated 630000 cases of MDR-TB among 12 millionprevalent cases of TB [1]The biggest challenge in any control

and treatment system for TB is MDR-TB It is estimated thatMDR-TB constitutes about 5 of all TB cases worldwide Asignificant parameter utilised to describe the phenotype ofdrug resistant strains of MTB including MDR strains is theenzymatic activity of the catalase-peroxidase system

The last decade has witnessed the development of theLAMP DNA amplification approach which can be appliedinstead of conventional PCR [2] and which offers the advan-tage of greater speed as well as being more sensitive specificand cost-effective [3 4] Once under way the reaction is ableto proceed on the basis of the strand displacement capabilityof the Bst DNA polymerase The temperature remains con-stant throughout and only one enzyme is required for the

Hindawi Publishing CorporationJournal of ChemistryVolume 2016 Article ID 3474396 8 pageshttpdxdoiorg10115520163474396

2 Journal of Chemistry

test Novel loop-mediated isothermal amplification (LAMP)can also detect drug resistance which is a one-pointmutationby designing an inner primer of 51015840 end specific mutant [5 6]

Nanoparticle-based assays represent a current trend inDNA detection [7] In particular Au-nanoparticles (AuNPs)have unique optical properties that make them suitable forthe design of labelled Au-nanoprobes They offer advantagesin that they are inexpensive and afford visual detection unlikemore conventional methods of detection such as those usedin fluorescence or radioactivity-based assays Previous workin colorimetric detection approaches using Au-nanoprobescoupled to LAMP has been described [8] Recently thecombination of LAMPamplification andAuNPprobes detec-tion has been developed successfully for detection of themutations associated with rifampicin resistance in MTB [9]However this experiment conducted an analysis in terms ofthe absorbance ratio and assay on the spectrophotometryso it would not be convenient for detection of INHr-TB infield samples The instant study demonstrated that LAMPamplification combinedwith theAu-nanoprobes system tookless than 75min to yield a colorimetric result observed fordetection of INHr-TB using a suitable eye detector

In this study the researchers designed a strategy basedon anAu-nanoprobe detection protocol combinedwith loop-mediated isothermal amplification (LAMP) for the rapid anddiscriminatory detection of MDR-TB and TB strains as wellas for the simultaneous characterisation of the presence ofmutations associated with isoniazid resistance In conductingfield analysis the speed and heightened specificity of LAMPalong with its isothermal profile make this approach idealfor DNA sample amplification in comparison with standardPCR This method is advantageous because it is swift inex-pensive specific and sensitive toMDR-TB detection It couldbe convenient for detection of MDR-TB in field samples aswell

2 Materials and Methods

21 Sample Preparation The 46 clinical samples were pro-vided by the Bureau of Tuberculosis which operates underthe Department of Disease Control Thai Ministry of PublicHealth All clinical samples were identified as katG genemutations associated with INH resistance by using GenotypeMDR-TB Plus (Hain Lifescience GmbH Nehren Germany)There were 31 samples of TB with INH resistance 15 sampleswith INH susceptibility and H37RV strain used as negativecontrol However Genotype MDR-TB Plus detection wasconfirmed after conventional methods detected cultures onLowenstein-Jensen (L-J) medium after 8 weeks After thatall samples were extracted DNA for LAMP amplificationusing a modified method from Rienthong et al [10] Brieflyone loopful of cells was first suspended in distilled water(300 120583L) before undergoing 20min of subsequent heat treat-ment at a temperature of 95∘C in Thermoblock Sonificationwas then performed for 15min using an ultrasonic bath atthe highest speed setting The samples were then sent toa standard centrifuge containing an aerosol-tight rotor for5min spinning at 10000timesg The LAMP amplification stageinvolved the use of a supernatant

22 Primers and Probe Designed Specific katG Gene of INHr-TB The LAMP primers for amplification of specific katGgenes in MTB were synthesised from Bio Basic Canada Incusing the manual designed and primer-designing softwarePrimer Explorer Ver 3 (Eiken Chemical Co Ltd) Theprimer sets consisted of outer primers (F3 and B3) and innerprimers (FIP and BIP) The directions and details for theprimers are shown in Table 1 and Figure 1

All oligonucleotide DNA probes and complementaryDNA targets for the detection of MDR-TB and MTB weredesigned based on the nucleotide sequence of katG generetrieved fromNCBI (Accession number X680811)The genehad 4810 base pairsNucleotide sequences at positions 2924ndash2938 (15 bp) were selected for synthesis of the DNA probeand specificity to the DNA target of MDR-TB and MTBFor DNA probe synthesis the linker of d(A

10) was added to

the 51015840 end with the thiol (Table 1) The accuracy of theprobewas confirmedusing a BLASTnucleotide-free programprovided by NCBI The probe was specific to MTB putativesecretion system-associated gene clusters This probe wasa thiol-modified oligonucleotide at the 51015840 end to form anSH group and gold atom with formation of an S-Au bondinteraction The thiol-labelling probe was synthesised fromBio Basic Canada Inc

23 LAMP Amplification of the Target katG Gene in M tuber-culosis A final volume of 25120583L was used for the reactionswith contents comprising 02mMof each of the outer primersF3 and B3 2mM of each of the inner primers FIP andBIP and also 08ndash20mM of dNTP mix (Promega MadisonWI USA) 1x of the supplied buffer (New England BiolabsInc Beverly MA USA) 06M betaine (Sigma-Aldrich StLouis MO USA) 4ndash10mM MgSO

4 25ndash50U of Bst 20

warm start DNA polymerase (large fragment New EnglandBiolabs Inc BeverlyMAUSA) and 1 120583L of template plasmidDNA (tenfold serial dilutions from 10minus0 to 10minus5 of totalDNA of MDR-TB and standard strain H37Rv) in a finalvolume of 25 120583L A reaction mixture containing distilledwater was used as negative control The reaction mixturewas incubated for 60min at 61∘C before subsequently under-going 2min of heating at 95∘C in order to deactivate theenzyme and thus cause the reaction to stop The resultingLAMP products were then stored at a temperature of 4∘CThey were analysed for LAMP products by 2 agarose gelelectrophoresis in a 05x Tris Borate EDTA (TBE) buffer at100 volts and observed under a Maestrogen Ultra Slim-LEDblue light transilluminator by MaestroSafe Nucleic Acid GelStain Sample (Maestrogen)

24 Gold-Nanoparticle Synthesis Prior to the preparation ofAuNP preparation the glassware involved underwent treat-ment using aqua regia (Sigma-Aldrich) in order to alleviatethe problems caused when AuNPs stick to the vial surfacesthereby lowering the effective concentrations in the vialsAuNPs were prepared by modifying a method used by LiuandYi [11] and Suebsing et al [12] Briefly 1mMhydrogen tre-trachloroaurate trihydrate (Sigma-Aldrich) was stirred andmixed in 250mL of distilled water while vigorously boilingprior to adding 388mM of sodium citrate tribasic dihydrate

Journal of Chemistry 3

2701

2761 AACAGCGGCGCTGATCGTCGGCGGTCACACTTTCGGTAAGACCCATGGCGCCGGCCCGGC

2821 CGATCTGGTCGGCCCCGAACCCGAGGCTGCTCCGCTGGAGCAGATGGGCTTGGGCTGGAA

2881 GAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATCACCA(GC)CGGCATCGAGGTCGTATG

2941

katG315 MT probe

GACGAACACCCCGACGAAATGGGACAACAGTTTCCTCGAGATCCTGTACGGCTACGAGTG-3998400

5998400-CATGGCCGCGGCGGTCGACATTCGCGAGACGTTTCGGCGCATGGCCATGAACGACGTCGA

F3-katGrarr

F2-katGrarr B1C-katGrarr

larrB2-katG

larr F1C-katG

larrB3-katG

GGCCGTAGCTCCAGC

Figure 1 The directions and details of LAMP primers for amplification of a specific katG gene ofM tuberculosis

Table 1 LAMP primers and Au-nanoprobe sequences

Primersprobe Nucleotide sequence 51015840 to 31015840 Numbers of base (bp)katG315 MT probe 51015840-Thiol-AAAAAAAAAACGACCTCGATGCCGG-31015840 15F3-katG 51015840-GAAACAGCGGCGCTGATC-31015840 18B3-katG 51015840-CGAGGAAACTGTTGTCCCAT-31015840 20FIP-katG 51015840-GTGGTGATCGCGTCCTTACCTTTTAGAGCTCGTATGGCACCGGAA-31015840 45BIP-katG 51015840-CTGGCATCGAGGTCGTATTTTTTCGTCGGGGTGTTCGTCC-31015840 40

(Sigma-Aldrich)The solution was subsequently permitted tocool until reaching room temperature The cooling phasesweremarked by a colour change in the solution starting fromyellow and then turning to clear before changing again toblack then purple and finally a deep redThe diameter of theAuNPs was about 15 nm with a surface plasmon band centreat 520 nm It was stored at 4∘C prior to use

All reagents and solvents were of the highest purity Milli-poreMilliQ water (18MΩcmminus1) was used in all experiments

25 Au-Nanoprobe Conjugated with a DNA Probe The thi-olated DNA probe was conjugated with Au-nanoparticlesusing a method reported by Mirkin et al [13] Initially 20120583Lof the 100 120583M thiolated DNA probe was incubated with 4mLof 10 nM Au-nanoparticles in a shaker at 100 rpm and 45∘Cfor 24 hours in the dark Then the solution was addedto 01M NaCl 10mM phosphate buffer (pH 7) and 10SDS Subsequently it was shaken at 100 rpm at 45∘C for 48hours It was centrifuged at 13000 rpm for 30min to removeexcess reagentsThe precipitate waswashed twicewith 500120583Lof resuspension buffer containing 10mM PBS (pH 74)150mM NaCl and 01 SDS after which it was resuspendedin 50120583L of the same buffer The fully functionalised DNA-Au-nanoparticle conjugations (Au-nanoprobes) retained thesame colour as the unmodified Au-nanoparticles with novisible aggregates It was stored at 4∘C until used

The Au-nanoprobe solution was confirmed by UVVisspectroscopy The peak for Au-nanoprobes was at 520 nmand its calculated concentration was the optimal amount fordetection which was approximately 108 nM

26 Optimal Hybridisation of Au-Nanoprobes with LAMPAmplicons For hybridisation diluted LAMP products from1 to 9 120583L each were mixed with the diluted Au-nanoprobescomplex in equal volume The mixture was hybridised at

61∘C for 10min After hybridisation 07MMgSO4was added

to the mixture in equal volume to induce aggregation of theAu-nanoprobes at room temperature for 5minThe solutionrsquoscolour change was discerned by direct observation Thecomplex solutions of Au-nanoprobeLAMP products weredetermined by UVvis spectra at 520 nm for confirmation

27 Optimal Concentration of MgSO4 for Detection by Colori-metric Assay Theoptimal concentration ofMgSO

4for detec-

tion by colorimetric assay was determined After hybridisa-tion 5 120583L of MgSO

4was added variously from 03 05 and

07M to themixture in equal volume to induce aggregation ofAu-nanoprobes at room temperature for 5minThe solutionrsquoscolour change was witnessed by direct observation

28 Detection by Colorimetric Assay Colorimetric assay wasperformed in a final volume of 15120583L containing a fixedvolume at 5 120583L of LAMP products from samples mixed with5 120583L of Au-nanoprobe complex The mixture was hybridisedat 61∘C for 10min After hybridisation 5 120583L of 07M MgSO

4

was added to the mixture to induce aggregation of Au-nanoprobes at room temperature for 5min The solutionrsquoscolour change was witnessed by direct observation Thecomplex solutions of Au-nanoprobeLAMP products weredetermined by UVvis spectra at 520 nm for confirmationThe positive samples were observed as being of a reddish-purple colour whereas the negative samples were observedas being of a bluish colour Water was used as negativecontrol If the water was observed to be reddish-purple theperformance of the system was considered invalid

29 Clinical Samples Test The forty-six clinical isolates usedwere obtained from tuberculosis cluster Bureau of AIDS-TB-STIs including 31 samples of TB with resistance to INH and15 susceptible to INH Additionally one strain of H37RV wasused as negative control Genotype MDR-TB Plus detection


was confirmed after conventional methods detected cultureson a Lowenstein-Jensen (L-J) medium after 8 weeks DNAwas extracted from cultures with the modified methodmentioned previously (Rienthong et al) and used for LAMPamplification The LAMP combined Au-nanoprobe assaysused for identification of 46 clinical samples in this studyweredetected individually using the colorimetric assay comparedto the standard Genotype MDR-TB Plus assays

3 Results

The authors created a two-stage approach with its foundationin both the molecular signatures of the MDR-TB membersand also the most widely encountered mutations related toINH resistance in MTB The key step of the process involvedthe LAMP amplification of a katG gene fragment followedby its hybridisation using specific Au-nanoprobes The katGlocus shared by all members of MDR-TB were targeted and aprobe specific to MDR-TB members was designed (katG315MT probe) One additional set of probes specific to the mostcommon point mutations associated with INH resistance(katG315) was also synthesised Each set was composed of thecomplement to the mutation

31 Optimal LAMP INHr-TB Reaction The researchersdetermined the optimal temperature for LAMP INHr-TBreaction to detect a specific katG gene of MDR-TB Theoptimisation of LAMP products detected in the optimumtemperature was carried out at 61∘C (Figure 2) and the opti-mal concentration ofMgSO

4was used for the LAMP reaction

to detect the specific katG gene of MDR-TB The LAMPreaction was carried out at 4mM MgSO

4 The optimum

concentration of dNTP for LAMP reaction was carried outat 16mM of concentration dNTP The LAMP detection ofMDR-TB showed a limited condition at 10minus3 dilution of theDNA templates (genomic DNA) for LAMP tests performedunder optimised conditions (Figure 3)

32 Optimal Concentration of MgSO4 for Detection by Colori-metric Assay MgSO

4concentration was used for detection

by colorimetric assay of a specific katG gene of MDR-TBThe positive samples of a reddish-purple colour (no colourchange) were formedwith 05 and 07MofMgSO

4(Figure 4)

For colorimetric assay the colorimetric reaction was carriedout with 07M of MgSO

4concentration and the colorimetric

reaction was followed with the concentration describedabove The solutionrsquos colour change was witnessed by directobservation

33 Optimal Hybridisation for Au-Nanoprobe Assay Theresearchers determined the optimal hybridisation of 5 120583LLAMP products with 5 120583L of Au-nanoprobe complex Themixturewas hybridised at 61∘C for 10minThen 5120583L of 07MMgSO

4was added to induce aggregation of Au-nanoprobes

at room temperature for 5min The solutionrsquos colour changewas observed directly (Figure 5) These conditions were usedfor the assay

34 Au-Nanoprobes Colorimetric Assay Solutions contain-ing the Au-nanoprobe revealed a red colouration due to

M MMut

STB

Neg

Mut

STB

Neg

Mut

Neg

Mut

STB

Neg

STB

500bp

59∘C 61∘C 63∘C 65∘C

Figure 2 Determining the optimal temperature for LAMP Lanes 1and 14 (M) Ladder DNA marker Lanes 2ndash13 reaction for 60minat 59 61 63 and 65∘C Mut as MDR-TB STB as H37RV Neg asnegative All LAMP products were electrophoresed on 2 agarosegels and stained with MaestroSafe Nucleic Acid Gel Stain Sample(Maestrogen)

MM

utST

BM

utST

BM

utST

BM

utST

BM

utST

BM

utST

BN

eg

500bp

100 10minus1 10minus2 10minus3 10minus4 10minus5

(a)

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Neg


(b)

Figure 3 Determining the DNA templates for LAMP of MDR-TB members via the Au-nanoprobes assay (a) Agarose gel showedLAMP products Lane 1 (M) Ladder DNAmarker Lanes 1ndash12 DNAtemplates for LAMP at 100ndash10minus5 Mut as MDR-TB STB as H37RVand Neg as negative All LAMP products were electrophoresed on2 agarose gels and stainedwithMaestroSafeNucleic AcidGel StainSample (Maestrogen) (b) Corresponding results recorded visuallyafter salt challenge Red colour showed the presence of the targetand purple indicated its absence

the localised surface plasmon resonance (SPR) of the Au-nanoprobes When the complementary target was presentthese Au-nanoprobes had protection from aggregationinduced by salt and therefore the solution did not changecolour However if a noncomplementary target was presentno protection existed and the solution duly changed to


Mut STB Mut STB Mut STB

03M MgSO4

5120583L05M MgSO4

5120583L07M MgSO4

5120583L

Figure 4 Determining the optimal concentration of MgSO4for

detection by colorimetric assay

Optimum condition

9 1

8 2

7 3

6 4

5 5

4 6

3 7

2 8

1 9

+5120583L 07M MgSO4

LAMP productsAu-katG315 probes (120583L)

Figure 5 Determining the optimal condition of hybridisation forAu-nanoprobe assay

blue (Figures 6(a)-6(b)) The data derived from UV-Visspectroscopy performed on the samples as shown in Fig-ure 6(c) support the hypothesis related to visual discrimi-nation of the sample being aggregation induced The samplewhich contained MDR-TB genomic DNA clearly revealed anabsorbance peak characteristic of Au-nanoprobes at 520 nmThis was because the free conduction band electrons ofthe dispersed particles become collectively excited In thecontrol samples andnegative samples the existence of a broadabsorbance spectrum indicated a peak shift denoting a longerwavelength (ge600 nm) as the particles in the aggregatesundergo coupling

35 Application of the LAMPCombined Au-Nanoprobes Assayfor INHr-TB Detection All clinical samples were used fordetection of the katG gene (31 positive samples and 15negative samples were confirmed by Genotype MDR-TBPlus) whichwere then assayed for positive sample presence ofpolymorphism in codons 315 of the katGgene (grarrc) (Figures7ndash10) The Au-nanoprobe results were compared to thoseobtained via Genotype MDR-TB Plus summary in Table 2The Au-nanoprobe assay showed sensitivity and specificity at100 in a test compared with Genotype MDR-TB Plus

4 Discussion

On the basis of the MDT-TB membersrsquo molecular signaturesalong with the most frequently observed mutations linkedwith INH resistance in M tuberculosis the authors estab-lished a two-stage process involving LAMP amplification of akatG gene fragment followed by hybridisation using specificAu-nanoprobes The katG locus shared by all members ofMDR-TB was targeted and a probe specific to INHr-TBmembers was designed (katG315 probe)

M Mut STB Neg

500bp

(a)

Mut STB Neg

(b)

0002004006008

01012014016018

02

400 450 500 550 600 650 700

Abso

rban

ce (O

D)

Wavelength (nm)

Mut positiveSTB controlNegative

(c)

Figure 6 Au-nanoprobe colorimetric assay (a) Agarose gel show-ing LAMP products Lane 1 M Ladder DNA marker Lane 2 Mut(MDR-TB) Lane 3 STB (Standard strain H37RV) and Lane 4 Neg(negative) (b) Corresponding results recorded visually after saltchallenge (c) UV-Vis spectra of the respective samples

In this study the researchers designed a strategy basedon the LAMP of DNA samples coupled to specific Au-nanoprobes It showed the potential to provide a rapid andspecific method for detection of isoniazid resistance (INHr)at katG gene position 315 (GrarrC) The researchers deter-mined the optimal temperature for LAMP reaction in thedetection of specific katG gene of INHr-TB at 61∘C for


STB

Neg

500bp

M 1 2 3 4 5 6 7 8

(a)

STB

Neg

1 2 3 4 5 6 7 8

(b)

Figure 7 Au-nanoprobe colorimetric assay for MDR-TB detection(a) Agarose gel showed LAMP products Lane M Ladder DNAmarker Lanes 1ndash8 Mut (MDR-TB) Lane STB standard strainH37RV and Lane Neg negative (b) Corresponding results recordedvisually after salt challenge A red colour showed the presence of thetarget while purple indicated its absence

Table 2MDR-TBmember detection using theAu-nanoprobe assayTest results from the Au-nanoprobes assay were subsequently com-pared with results from the Genotype MDR-TB Plus assay

GenotypeMDRTB PlusTB assay

Au-nanoprobes assay kat G315 probePositive Negative Total

INH (315) resistant (31) 31 0 31INH susceptible (15) 0 15 15Total (46) 31 15 46

60min The MgSO4and dNTP concentrations were opti-

mised because they were able to influence LAMP reactionsas described previously [2] It was also found that at least4mM MgSO

4was required in the LAMP reaction although

inhibition could result in higher than 10mM MgSO4due to

reduced activity of the Bst 20 warm start DNA polymeraseand destabilisation of the DNA helix [2] The dNTP mixat 08mM was minimal for the LAMP reaction under theoptimal MgSO

4concentration The researchers found the

optimal condition for LAMP reaction at 4ndash6mM MgSO4

08ndash16mM dNTP mix and 25ndash50U of Bst 20 warm startDNA polymerase to be suitable for detection of drug resis-tance which is a one-point mutation associated with INHrin MTB The determination of DNA templates for LAMPof MDR-TB members was via the Au-nanoprobe assay The

STB

Neg

500bp

M 9 10 11 12 13 14 15 16

(a)

STB

Neg

9 10 11 12 13 14 15 16

(b)

Figure 8 Au-nanoprobe colorimetric assay for MDR-TB detection(a) Agarose gel showed LAMP products Lane M Ladder DNAmarker Lanes 9ndash16 Mut (MDR-TB) Lane STB standard strainH37RV and Lane Neg negative (b) Corresponding results recordedvisually after salt challenge Red colour showed the presence of thetarget while purple indicated its absence

Mut Sus

M

500bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(a)

Mut Sus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(b)

Figure 9 Detection of MDR-TBmembers via Au-nanoprobe assay(a) Agarose gel showed LAMP products Lane 1 (M) Ladder DNAmarker Lanes 1ndash16 Mut as MDR-TB Sus as INH susceptible andNeg as negative (b) Corresponding results recorded visually aftersalt challenge Red colour showed the presence of the target whilepurple indicated its absence


Mut Sus

M Neg

500bp

17 18 19 20 21 22 23 24 25 26 27 28 29 30

(a)

Mut Sus

Neg17 18 19 20 21 22 23 24 25 26 27 28 29 30

(b)

Figure 10 Detection ofMDR-TBmembers via Au-nanoprobe assay(a) Agarose gel showed LAMP products Lane 1 (M) Ladder DNAmarker Lanes 17ndash30 Mut as MDR-TB Sus as INH susceptibleand Neg as negative (b) Corresponding results recorded visuallyafter salt challenge Red colour showed the presence of the targetwhile purple indicated its absence

LAMP detection of MDR-TB showed a limited conditionat 10minus3 dilution of the DNA templates (genomic DNA) forLAMP tests performed under optimised conditions Thiscorresponded to the detection of DNA template limits forLAMP of MDR-TB members via the Au-nanoprobe assay

Specific hybridisation between the LAMP ampliconscombined with Au-nanoprobes resulted in the formation ofa three-dimensional polymeric network of non-cross-linkedAu-nanoprobes that prevented them from forming closeaggregates Thus they maintained their deep red colourationwhen salt was present However when the Au-nanoprobeswere left unprotected aggregation took place when salt waspresent in the blank and in the negative control whichcontained the noncomplementary DNA The absorbancespectrum consequently experienced a quantum shift whichcould be observed in the form of a colour change to bluefrom the original red [8] However this method was 2-stepLAMP test we must open the cover of the reaction tube inorder to add Au-nanoprobes In this time there is the risk ofthe contamination of the LAMP products in other samplesFor this reason the Au-nanoprobe reacted with LAMPamplicons contamination could not overcome negative resultthat contamination is a rare event Moreover the LAMPamplification inactivated the enzyme by heating at 95∘C for2min in terminating the reaction The optimisation testsrevealed the importance of the ratio between LAMP ampli-cons and Au-nanoprobes since this would influence whetherthe detection of hybridisation between Au-nanoprobes andLAMPampliconswould succeed and alsowhether itmight be

reproducibleThemost suitable ratio was shown to be 5 5 120583LThe salt concentration (5120583L of 07mM MgSO

4) used to

promote aggregation after the hybridisation step was criticalas well In addition to colorimetric reaction confirmation ofthe LAMP productsAu-nanoprobe complex solutions wasdetermined by UVvis spectra at 520 nm for confirmation

The Au-nanoprobe system proposed in this study wascapable of detecting MDR-TB members and mutations asso-ciated with isoniazid resistance while also being easy to per-form without the need for expensive and complex laboratoryset-ups previously studied [14] It is important that theMDR-TB members and mutations within the katG geneshould be quickly and accurately identified since this aidsthe predication at a high confidence level of whether or notthe strain under examination is MDR-TB

In summary the researchers designed a strategy based onAu-nanoprobe detection protocol combined with LAMP forthe rapid detection of MDR-TB strains as well as simultane-ous characterisation of the presence of mutations associatedwith isoniazid resistance In field analysis it was shown thatthe application of LAMP to carry out the DNA sample ampli-fication was highly appropriate since the greater specificityand speed along with the isothermal profile resulted in amore effective approach in comparison with standard PCRThis method was advantageous because it was quick cheapspecific and sensitive for MDR-TB detection It could beconvenient for the detection of MDR-TB in field samples aswell

5 Conclusions

The goal of creating simple but robust portable platformsfor molecular diagnosis has attracted considerable attentionsince it will support the fight against TB in peripheral lab-oratories and at point-of-care facilities The Au-nanoprobesystem proposed in this research was capable of detecting notonly MDR-TB members but also mutations which are linkedwith isoniazid resistance (INHr) It offered the advantageof being simple to use and did not require expensive well-equipped laboratory facilities MDR-TB members and muta-tions within the katG gene could be quickly and accuratelyidentified which represents a significant advantage since itallows predictions regarding the identity of a strain to bemade with a higher degree of confidence This ensuresthat patients carrying MDR-TB can be quickly diagnosedisolated and treated thereby reducing the problem of non-compliance In tests it was shown that in comparison withthe Genotype MDR-TB Plus TB assay Au-nanoprobes wereable to correctly confirm the presenceMDR-TBDNA in 100of samples Following LAMP amplification the combinedAu-nanoprobe system took less than 75min to yield a colori-metric result using a suitable eye detector In addition testswere carried out to assess the potential use through a portableassay platform confirming the suitability of the approach forfield screening Further studies may be required in order tooptimise the validation methodology and to determine thebest approach for the direct application to clinical samplesThe concept should also be extended to be applied in the caseof othermutations which have been linked to drug resistance


Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by Srinakharinwirot UniversityAdditionally the researchers would like to acknowledge theassistance provided by the Bureau of Tuberculosis in theThaiMinistry of Public Health

References

[1] WorldHealth Organisation ldquoGlobal tuberculosis controlrdquo 2012httpwwwwhointtbpublicationsglobal reportenindexhtml

[2] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 no 12 article E63 2000

[3] S Ikeda K Takabe M Inagaki N Funakoshi and K SuzukildquoDetection of gene point mutation in paraffin sections using insitu loop-mediated isothermal amplificationrdquo Pathology Inter-national vol 57 no 9 pp 594ndash599 2007

[4] Y Mori K Nagamine N Tomita and T Notomi ldquoDetectionof loop-mediated isothermal amplification reaction by turbidityderived frommagnesium pyrophosphate formationrdquo Biochemi-cal and Biophysical Research Communications vol 289 no 1 pp150ndash154 2001

[5] M Iwasaki T Yonekawa K Otsuka et al ldquoValidation ofthe loop-mediated isothermal amplification method for sin-gle nucleotide polymorphism genotyping with whole bloodrdquoGenome Letters vol 2 no 3 pp 119ndash126 2003

[6] A Badolo K Okado W M Guelbeogo et al ldquoDevelopmentof an allele-specific loop-mediated isothermal amplificationmethod (AS-LAMP) to detect the L1014F kdr-w mutation inAnopheles gambiae s lrdquoMalaria Journal vol 11 article 227 2012

[7] P V Baptista E Pereira P Eaton et al ldquoGold nanoparticles forthe development of clinical diagnosis methodsrdquo Analytical andBioanalytical Chemistry vol 391 no 3 pp 943ndash950 2008

[8] T Kaewphinit S Santiwatanakul and K Chansiri ldquoColori-metric DNA based biosensor combined with loop-mediatedisothermal amplification for detection ofMycobacterium tuber-culosis by using gold nanoprobe aggregationrdquo Sensors amp Trans-ducers vol 142 pp 123ndash128 2013

[9] B Veigas P Pedrosa I Couto M Viveiros and P V BaptistaldquoIsothermal DNA amplification coupled to Au-nanoprobes fordetection of mutations associated to Rifampicin resistance inMycobacterium tuberculosisrdquo Journal of Nanobiotechnology vol11 no 1 article 38 2013

[10] D Rienthong S Rienthong C Boonin S Woraswad and YKasetjaroen ldquoRapid detection for early appearance of rifampinand isoniazid resistance in Mycobacterium tuberculosisrdquo SirirajMedical Journal vol 61 pp 49ndash55 2009

[11] J Liu and L Yi ldquoPreparation of aptamer-linked gold nanopar-ticle purple aggregates for colorimetric sensing of analytesrdquoNature Protocols vol 1 no 1 pp 246ndash252 2006

[12] R Suebsing P Prombun J Srisala and W KiatpathomchaildquoLoop-mediated isothermal amplification combined with col-orimetric nanogold for detection of the microsporidian Ente-rocytozoon hepatopenaei in penaeid shrimprdquo Journal of AppliedMicrobiology vol 114 no 5 pp 1254ndash1263 2013

[13] C A Mirkin R L Letsinger R C Mucic and J J StorhoffldquoA DNA-based method for rationally assembling nanoparticlesintomacroscopic materialsrdquoNature vol 382 no 6592 pp 607ndash609 1996

[14] B Veigas D Machado J Perdigao et al ldquoAu-nanoprobesfor detection of SNPs associated with antibiotic resistance inMycobacterium tuberculosisrdquo Nanotechnology vol 21 no 41Article ID 415101 2010

Submit your manuscripts athttpwwwhindawicom

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Inorganic ChemistryInternational Journal of

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test Novel loop-mediated isothermal amplification (LAMP)can also detect drug resistance which is a one-pointmutationby designing an inner primer of 51015840 end specific mutant [5 6]

Nanoparticle-based assays represent a current trend inDNA detection [7] In particular Au-nanoparticles (AuNPs)have unique optical properties that make them suitable forthe design of labelled Au-nanoprobes They offer advantagesin that they are inexpensive and afford visual detection unlikemore conventional methods of detection such as those usedin fluorescence or radioactivity-based assays Previous workin colorimetric detection approaches using Au-nanoprobescoupled to LAMP has been described [8] Recently thecombination of LAMPamplification andAuNPprobes detec-tion has been developed successfully for detection of themutations associated with rifampicin resistance in MTB [9]However this experiment conducted an analysis in terms ofthe absorbance ratio and assay on the spectrophotometryso it would not be convenient for detection of INHr-TB infield samples The instant study demonstrated that LAMPamplification combinedwith theAu-nanoprobes system tookless than 75min to yield a colorimetric result observed fordetection of INHr-TB using a suitable eye detector

In this study the researchers designed a strategy basedon anAu-nanoprobe detection protocol combinedwith loop-mediated isothermal amplification (LAMP) for the rapid anddiscriminatory detection of MDR-TB and TB strains as wellas for the simultaneous characterisation of the presence ofmutations associated with isoniazid resistance In conductingfield analysis the speed and heightened specificity of LAMPalong with its isothermal profile make this approach idealfor DNA sample amplification in comparison with standardPCR This method is advantageous because it is swift inex-pensive specific and sensitive toMDR-TB detection It couldbe convenient for detection of MDR-TB in field samples aswell

2 Materials and Methods

21 Sample Preparation The 46 clinical samples were pro-vided by the Bureau of Tuberculosis which operates underthe Department of Disease Control Thai Ministry of PublicHealth All clinical samples were identified as katG genemutations associated with INH resistance by using GenotypeMDR-TB Plus (Hain Lifescience GmbH Nehren Germany)There were 31 samples of TB with INH resistance 15 sampleswith INH susceptibility and H37RV strain used as negativecontrol However Genotype MDR-TB Plus detection wasconfirmed after conventional methods detected cultures onLowenstein-Jensen (L-J) medium after 8 weeks After thatall samples were extracted DNA for LAMP amplificationusing a modified method from Rienthong et al [10] Brieflyone loopful of cells was first suspended in distilled water(300 120583L) before undergoing 20min of subsequent heat treat-ment at a temperature of 95∘C in Thermoblock Sonificationwas then performed for 15min using an ultrasonic bath atthe highest speed setting The samples were then sent toa standard centrifuge containing an aerosol-tight rotor for5min spinning at 10000timesg The LAMP amplification stageinvolved the use of a supernatant

22 Primers and Probe Designed Specific katG Gene of INHr-TB The LAMP primers for amplification of specific katGgenes in MTB were synthesised from Bio Basic Canada Incusing the manual designed and primer-designing softwarePrimer Explorer Ver 3 (Eiken Chemical Co Ltd) Theprimer sets consisted of outer primers (F3 and B3) and innerprimers (FIP and BIP) The directions and details for theprimers are shown in Table 1 and Figure 1

All oligonucleotide DNA probes and complementaryDNA targets for the detection of MDR-TB and MTB weredesigned based on the nucleotide sequence of katG generetrieved fromNCBI (Accession number X680811)The genehad 4810 base pairsNucleotide sequences at positions 2924ndash2938 (15 bp) were selected for synthesis of the DNA probeand specificity to the DNA target of MDR-TB and MTBFor DNA probe synthesis the linker of d(A

10) was added to

the 51015840 end with the thiol (Table 1) The accuracy of theprobewas confirmedusing a BLASTnucleotide-free programprovided by NCBI The probe was specific to MTB putativesecretion system-associated gene clusters This probe wasa thiol-modified oligonucleotide at the 51015840 end to form anSH group and gold atom with formation of an S-Au bondinteraction The thiol-labelling probe was synthesised fromBio Basic Canada Inc

23 LAMP Amplification of the Target katG Gene in M tuber-culosis A final volume of 25120583L was used for the reactionswith contents comprising 02mMof each of the outer primersF3 and B3 2mM of each of the inner primers FIP andBIP and also 08ndash20mM of dNTP mix (Promega MadisonWI USA) 1x of the supplied buffer (New England BiolabsInc Beverly MA USA) 06M betaine (Sigma-Aldrich StLouis MO USA) 4ndash10mM MgSO

4 25ndash50U of Bst 20

warm start DNA polymerase (large fragment New EnglandBiolabs Inc BeverlyMAUSA) and 1 120583L of template plasmidDNA (tenfold serial dilutions from 10minus0 to 10minus5 of totalDNA of MDR-TB and standard strain H37Rv) in a finalvolume of 25 120583L A reaction mixture containing distilledwater was used as negative control The reaction mixturewas incubated for 60min at 61∘C before subsequently under-going 2min of heating at 95∘C in order to deactivate theenzyme and thus cause the reaction to stop The resultingLAMP products were then stored at a temperature of 4∘CThey were analysed for LAMP products by 2 agarose gelelectrophoresis in a 05x Tris Borate EDTA (TBE) buffer at100 volts and observed under a Maestrogen Ultra Slim-LEDblue light transilluminator by MaestroSafe Nucleic Acid GelStain Sample (Maestrogen)

24 Gold-Nanoparticle Synthesis Prior to the preparation ofAuNP preparation the glassware involved underwent treat-ment using aqua regia (Sigma-Aldrich) in order to alleviatethe problems caused when AuNPs stick to the vial surfacesthereby lowering the effective concentrations in the vialsAuNPs were prepared by modifying a method used by LiuandYi [11] and Suebsing et al [12] Briefly 1mMhydrogen tre-trachloroaurate trihydrate (Sigma-Aldrich) was stirred andmixed in 250mL of distilled water while vigorously boilingprior to adding 388mM of sodium citrate tribasic dihydrate


2701




2941

katG315 MT probe



F3-katGrarr


larrB2-katG

larr F1C-katG

larrB3-katG

GGCCGTAGCTCCAGC












4for detec-





4





3 Results





4 The optimum





4(Figure 4)








M MMut

STB

Neg

Mut

STB

Neg

Mut

Neg

Mut

STB

Neg

STB

500bp

59∘C 61∘C 63∘C 65∘C


MM

utST

BM

utST

BM

utST

BM

utST

BM

utST

BM

utST

BN

eg

500bp


(a)

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Neg


(b)





03M MgSO4

5120583L05M MgSO4

5120583L07M MgSO4

5120583L



Optimum condition

9 1

8 2

7 3

6 4

5 5

4 6

3 7

2 8

1 9

+5120583L 07M MgSO4





4 Discussion


M Mut STB Neg

500bp

(a)

Mut STB Neg

(b)

0002004006008

01012014016018

02

400 450 500 550 600 650 700

Abso

rban

ce (O

D)

Wavelength (nm)


(c)




STB

Neg

500bp

M 1 2 3 4 5 6 7 8

(a)

STB

Neg

1 2 3 4 5 6 7 8

(b)














STB

Neg

500bp

M 9 10 11 12 13 14 15 16

(a)

STB

Neg

9 10 11 12 13 14 15 16

(b)


Mut Sus

M

500bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(a)

Mut Sus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(b)



Mut Sus

M Neg

500bp

17 18 19 20 21 22 23 24 25 26 27 28 29 30

(a)

Mut Sus

Neg17 18 19 20 21 22 23 24 25 26 27 28 29 30

(b)





4) used to




5 Conclusions



Competing Interests


Acknowledgments


References
























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


2701




2941

katG315 MT probe



F3-katGrarr


larrB2-katG

larr F1C-katG

larrB3-katG

GGCCGTAGCTCCAGC












4for detec-





4





3 Results





4 The optimum





4(Figure 4)








M MMut

STB

Neg

Mut

STB

Neg

Mut

Neg

Mut

STB

Neg

STB

500bp

59∘C 61∘C 63∘C 65∘C


MM

utST

BM

utST

BM

utST

BM

utST

BM

utST

BM

utST

BN

eg

500bp


(a)

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Neg


(b)





03M MgSO4

5120583L05M MgSO4

5120583L07M MgSO4

5120583L



Optimum condition

9 1

8 2

7 3

6 4

5 5

4 6

3 7

2 8

1 9

+5120583L 07M MgSO4





4 Discussion


M Mut STB Neg

500bp

(a)

Mut STB Neg

(b)

0002004006008

01012014016018

02

400 450 500 550 600 650 700

Abso

rban

ce (O

D)

Wavelength (nm)


(c)




STB

Neg

500bp

M 1 2 3 4 5 6 7 8

(a)

STB

Neg

1 2 3 4 5 6 7 8

(b)














STB

Neg

500bp

M 9 10 11 12 13 14 15 16

(a)

STB

Neg

9 10 11 12 13 14 15 16

(b)


Mut Sus

M

500bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(a)

Mut Sus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(b)



Mut Sus

M Neg

500bp

17 18 19 20 21 22 23 24 25 26 27 28 29 30

(a)

Mut Sus

Neg17 18 19 20 21 22 23 24 25 26 27 28 29 30

(b)





4) used to




5 Conclusions



Competing Interests


Acknowledgments


References
























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



3 Results





4 The optimum





4(Figure 4)








M MMut

STB

Neg

Mut

STB

Neg

Mut

Neg

Mut

STB

Neg

STB

500bp

59∘C 61∘C 63∘C 65∘C


MM

utST

BM

utST

BM

utST

BM

utST

BM

utST

BM

utST

BN

eg

500bp


(a)

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Mut

STB

Neg


(b)





03M MgSO4

5120583L05M MgSO4

5120583L07M MgSO4

5120583L



Optimum condition

9 1

8 2

7 3

6 4

5 5

4 6

3 7

2 8

1 9

+5120583L 07M MgSO4





4 Discussion


M Mut STB Neg

500bp

(a)

Mut STB Neg

(b)

0002004006008

01012014016018

02

400 450 500 550 600 650 700

Abso

rban

ce (O

D)

Wavelength (nm)


(c)




STB

Neg

500bp

M 1 2 3 4 5 6 7 8

(a)

STB

Neg

1 2 3 4 5 6 7 8

(b)














STB

Neg

500bp

M 9 10 11 12 13 14 15 16

(a)

STB

Neg

9 10 11 12 13 14 15 16

(b)


Mut Sus

M

500bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(a)

Mut Sus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(b)



Mut Sus

M Neg

500bp

17 18 19 20 21 22 23 24 25 26 27 28 29 30

(a)

Mut Sus

Neg17 18 19 20 21 22 23 24 25 26 27 28 29 30

(b)





4) used to




5 Conclusions



Competing Interests


Acknowledgments


References
























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



03M MgSO4

5120583L05M MgSO4

5120583L07M MgSO4

5120583L



Optimum condition

9 1

8 2

7 3

6 4

5 5

4 6

3 7

2 8

1 9

+5120583L 07M MgSO4





4 Discussion


M Mut STB Neg

500bp

(a)

Mut STB Neg

(b)

0002004006008

01012014016018

02

400 450 500 550 600 650 700

Abso

rban

ce (O

D)

Wavelength (nm)


(c)




STB

Neg

500bp

M 1 2 3 4 5 6 7 8

(a)

STB

Neg

1 2 3 4 5 6 7 8

(b)














STB

Neg

500bp

M 9 10 11 12 13 14 15 16

(a)

STB

Neg

9 10 11 12 13 14 15 16

(b)


Mut Sus

M

500bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(a)

Mut Sus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(b)



Mut Sus

M Neg

500bp

17 18 19 20 21 22 23 24 25 26 27 28 29 30

(a)

Mut Sus

Neg17 18 19 20 21 22 23 24 25 26 27 28 29 30

(b)





4) used to




5 Conclusions



Competing Interests


Acknowledgments


References
























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


STB

Neg

500bp

M 1 2 3 4 5 6 7 8

(a)

STB

Neg

1 2 3 4 5 6 7 8

(b)














STB

Neg

500bp

M 9 10 11 12 13 14 15 16

(a)

STB

Neg

9 10 11 12 13 14 15 16

(b)


Mut Sus

M

500bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(a)

Mut Sus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Neg

(b)



Mut Sus

M Neg

500bp

17 18 19 20 21 22 23 24 25 26 27 28 29 30

(a)

Mut Sus

Neg17 18 19 20 21 22 23 24 25 26 27 28 29 30

(b)





4) used to




5 Conclusions



Competing Interests


Acknowledgments


References
























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Mut Sus

M Neg

500bp

17 18 19 20 21 22 23 24 25 26 27 28 29 30

(a)

Mut Sus

Neg17 18 19 20 21 22 23 24 25 26 27 28 29 30

(b)





4) used to




5 Conclusions



Competing Interests


Acknowledgments


References
























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Competing Interests


Acknowledgments


References
























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Development of Au-Nanoprobes Combined ...downloads.hindawi.com/journals/jchem/2016/3474396.pdf · Isoniazid Resistance in Mycobacterium tuberculosis JutturongCkumdee,