research article high resolution melting analysis for rapid...

Research ArticleHigh Resolution Melting Analysis for RapidMutation Screening in Gyrase and Topoisomerase IV Genes inQuinolone-Resistant Salmonella enterica

Soo Tein Ngoi12 and Kwai Lin Thong12

1 Institute of Biological Sciences Faculty of Science University of Malaya 50603 Kuala Lumpur Malaysia2 Laboratory of Biomedical Science and Molecular Microbiology Institute of Graduate Studies University of Malaya50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Kwai LinThong thongklumedumy

Received 8 June 2014 Accepted 19 September 2014 Published 12 October 2014

Academic Editor Stefano DrsquoAmelio

Copyright copy 2014 S T Ngoi and K L Thong 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

The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian regionThe objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes DNA sequencing was performed on 62 Salmonellastrains to identify mutations in the QRDR of gyrA gyrB parC and parE genes Mutations were detected in QRDR of gyrA (119899 = 52S83F S83Y S83I D87G D87Y and D87N) and parE (119899 = 1 M438I) Salmonella strains with mutations within QRDR of gyrA aregenerally more resistant to nalidixic acid (MIC 16 gt 256 120583gmL) Mutations were uncommon within the QRDR of gyrB parCand parE genes In the HRM assay mutants can be distinguished from the wild-type strains based on the transition of melt curveswhich is more prominent when the profiles are displayed in difference plot In conclusion HRM analysis allows for rapid screeningformutations at theQRDRs of gyrase and topoisomerase IV genes in SalmonellaThis assaymarkedly reduced the sequencing effortinvolved in mutational studies of quinolone-resistance genes

1 Introduction

Salmonella is a human pathogen commonly found indeveloped and developing countries causing clinical dis-eases ranging from mild gastroenteritis to septicaemia [1]Quinolones are broad spectrum antimicrobial agents thatinhibit bacterial DNA from unwinding and duplicatingduring cell division and are commonly used in treatingSalmonella infection However the worldwide emergence ofquinolone-resistant bacterial strains raises public health con-cern Increased incidence of quinolone-resistant Salmonellaserovars has been documented in Southeast Asian region [2ndash5]

Three major mechanisms contribute to quinolone-resistance among Salmonella altered protein targets forquinolones decreased uptake of quinolones by bacteriaand DNA gyrase protection via plasmid-derived qnr genes

[6] DNA gyrase and topoisomerase IV are two importantenzymes involved in bacterial DNA replication Quinolonesbind to gyrasetopoisomerase IV-DNA complex and inhibitDNA replication This action is responsible for the bacterio-static and bactericidal property of quinolones Mutations inthe bacterial genes encoding DNA gyrase and topoisomeraseIVmay confer resistance to quinolones and it has been shownthat altered structures of these enzymes prevent binding ofquinolones [6 7]

Both DNA gyrase and topoisomerase IV are tetramericenzymes DNA gyrase is encoded by gyrA and gyrB geneswhile topoisomerase IV is encoded by parC and parE genesMutation in quinolone-resistant determining region (QRDR)in these genes is associated with quinolone-resistance [6]Many studies have reported the presence of such muta-tions in quinolone-resistant Salmonella [8ndash12] Indeed lowerfluoroquinolone susceptibility and quinolone-resistance are

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 718084 8 pageshttpdxdoiorg1011552014718084

2 BioMed Research International

linked to point mutations in QRDR of the gyrase genes[13 14] While most studies revealed that codon 87 of gyrAgene represents the most frequently mutated amino acid inSalmonella [8 12 14] point mutations outside of QRDR mayalso play a role in quinolone-resistance [6] NeverthelessQRDR remains the major focus in quinolone-resistancestudies since this region exhibits high mutation rate

DNA sequencing is the gold standard for detectinggenetic changes However it is relatively costly to sequenceall four genes when examining a large sample pool Fur-thermore mutations are rare in parC and parE genes [9]therefore DNA sequencing will not be an economicallyviable option when dealing with such conserved regionsThereal-time polymerase chain reaction- (PCR-) based high-resolution melt curve (HRM) analysis is able to detect smallgenetic variation in PCR amplicons [15ndash17] This methodis highly sensitive and specific and has been employed insingle nucleotide polymorphism (SNP) scanning [18]DuringHRM analysis a DNA intercalating dye is included in thePCR reaction mixture This compound interacts specificallywith double-stranded DNA and emits fluorescence signalbut loses fluorescence when released from the DNA duringdenaturationUpon completion of PCR theDNAsamples aresubject to a temperature gradient and the loss of fluorescencesignal resulting from denaturation gives each DNA samplea unique melting curve that is detected by the real-timePCR system [19] Hence different Salmonella strains can bediscriminated based on their sequence length GC contentor strand composition

HRM assay has been used for the identification ofquinolone-resistance strains in pathogens such as Mycobac-terium tuberculosis Bacillus anthracis Yersinia pestis Fran-cisella tularensis and Salmonella Typhi and Paratyphi A [20ndash23] In these studies only gyrA gene was examined Althoughinfrequent mutations in gyrB parC and parE genes ofSalmonella and their correlation with lower susceptibility toquinolones and fluoroquinolones have been documented too[9] The objective of the study was to determine the potentialof HRM analysis for rapid screening of mutations in QRDRof Salmonella gyrase and topoisomerase IV genes

2 Materials and Methods

21 Bacterial Strains A total of 195 Salmonella enterica strainswere obtained from the culture collection of Laboratory ofBiomedical Sciences and Molecular Microbiology Instituteof Graduate Studies University of Malaya The susceptibilityof the strains towards nalidixic acid and ciprofloxacin wasexamined and reported in previous studies [24 25] Sixty-two Salmonella strains showing resistance or reduced sus-ceptibility towards nalidixic acid were used to develop theHRM assay in this study (Salmonella Typhimurium 119899 =12 Salmonella Enteritidis 119899 = 50) All selected strainswere susceptible to ciprofloxacin The minimum inhibitoryconcentration (MIC) of nalidixic acid for the strains wasdetermined by using Etest strips (BioMerieux Marcy lrsquoEtoileFrance) The test was performed according to manufacturerrsquosinstructions Genomic DNA for each bacterial strain was

extracted by using Wizard genomic DNA purification kit(Promega Madison USA) The details of all Salmonellastrains used in this study are listed in the SupplementaryTable (see Table S1 in the Supplementary Material availableonline at httpdxdoiorg1011552014718084)

22 DNA Sequencing PCR amplification of gyrA gyrBparC and parE QRDRs was performed using previouslydescribed primers [26] The selected primers amplify theQRDRs of the respective genes PCR amplification wasdone in a 25 120583L monoplex reaction mixture containing1x colourless GoTaq Flexi Buffer 15mmolL magnesiumchloride (MgCl

2) 200120583molL deoxynucleoside triphosphate

(dNTP) mix 03 120583molL of each primer pair 1 U Taq DNApolymerase (Promega Madison USA) and approximately100 ng of bacterial genomicDNAThe PCR reactionmixtureswere first incubated at 94∘C for 2min followed by 25 cycles of94∘C for 30 s 60ndash63∘C (annealing temperature varies for eachgene) for 30 s and 72∘C for 45 s with a final extension step of72∘C for 5min The appropriate annealing temperatures forgyrA gyrB parC and parE are 60∘C 60∘C 63∘C and 62∘Crespectively Agarose gel electrophoresis was then carriedout to confirm the presence of amplicons prior to DNAsequencing Next the PCR products were purified usingWizard SV gel and PCR clean-up system (PromegaMadisonUSA) and the purified PCR products were sent to First BASELaboratories (Selangor Malaysia) for sequencing Mutationsin the target genes were determined by comparison withthe wild-type sequence of the respective genes in SalmonellaTyphimuriumLT2 (GenBank accession numberNC 003197)

23 HRMAnalysis A homology search for gyrA gyrB parCand parE gene sequences of Salmonella serovars from theNCBI GenBank database was performed Sequence align-ment was done using the molecular evolutionary geneticsanalysis software version 5 (MEGA5) [27] Primers weredesigned using the online tool Primer3Plus (httpprimer3-pluscomcgi-bindevprimer3pluscgi) spanning the QRDRof each gene of interest and were commercially synthe-sized (NHK Bioscience Solutions Kuala Lumpur Malaysia)Approximately 20 ng of bacterial genomic DNA was addedinto 1x MeltDoctor HRM master mix (Applied BiosystemsFoster City CA USA) which was premixed with 03120583Mof each primer pair Sterile deionized distilled water wasadded to make up a final reaction volume of 20 120583L Thereproducibility of the assay was determined by performingall reactions in triplicate In each assay the DNA samplefrom awild-type reference strain was used as positive controlThe selection of the wild-type reference strains for thedifferent target genes was explained in the Results section(Section 32) Negative control for the experiment was theHRM reaction mix without the addition of bacterial DNAThe HRM assay was performed on the 7500 Fast Real-Time PCR System (Applied Biosystems) The real-time PCRamplification included a holding stage at 95∘C for 10minfollowed by the cycling stage with 40 cycles of 95∘C for 15 secand subsequently 60∘C for 1min The subsequent melt curvestage consisted of the following steps 95∘C for 15 sec a melt

BioMed Research International 3

Table 1 Mutations in the gyrase and topoisomerase IV genes as determined by DNA sequencing and the nalidixic acid minimum inhibitoryconcentration (MIC) of their corresponding Salmonella strains

Strain MIC(120583gmL) gyrAa gyrB parC parEa

SE 10907 gt256 D87Y Silent mutations None V521FSE 00503 gt256 D87Y Silent mutations None Silent mutationsSE 02203 gt256 D87Y Silent mutations None Silent mutationsSE 02603 gt256 D87Y Silent mutations None Silent mutationsSE 02703 gt256 D87Y Silent mutations None Silent mutationsSE 02803 gt256 D87Y Silent mutations None Silent mutationsSE 03203 gt256 D87Y Silent mutations None Silent mutationsSE 03303 gt256 D87Y Silent mutations None Silent mutationsSE 04704 gt256 D87Y Silent mutations None Silent mutationsSE 05405 gt256 D87Y Silent mutations None Silent mutationsSE 06405 gt256 D87Y Silent mutations None Silent mutationsSE 06605 gt256 D87Y Silent mutations None Silent mutationsSE 06705 gt256 D87Y Silent mutations None Silent mutationsSE 06905 gt256 D87Y Silent mutations None Silent mutationsSE 07305 gt256 D87Y Silent mutations None Silent mutationsSE 07505 gt256 D87Y Silent mutations None Silent mutationsSE 07805 gt256 D87Y Silent mutations None Silent mutationsSE 08005 gt256 D87Y Silent mutations None Silent mutationsSE 08105 gt256 D87Y Silent mutations None Silent mutationsSE 08606 gt256 D87Y Silent mutations None Silent mutationsSE 10107 gt256 D87Y Silent mutations None Silent mutationsSE 10407 gt256 D87Y Silent mutations None Silent mutationsSE 10507 gt256 D87Y Silent mutations None Silent mutationsSE 10607 gt256 D87Y Silent mutations None Silent mutationsSE 10707 gt256 D87Y Silent mutations None Silent mutationsSE 00203 192 D87Y Silent mutations None Silent mutationsSE 07605 192 D87Y Silent mutations None Silent mutationsSE 00403 128 D87Y Silent mutations None Silent mutationsSE 04804 128 D87Y Silent mutations None Silent mutationsSE 09706 128 D87Y Silent mutations None Silent mutationsSE 07405 96 D87Y Silent mutations None Silent mutationsSE 09006 96 D87Y Silent mutations None Silent mutationsSE 06805 64 D87Y Silent mutations None Silent mutationsSE 10207 64 D87Y Silent mutations None Silent mutationsSE 04604 48 D87Y Silent mutations None Silent mutationsSE 04904 48 D87Y Silent mutations None Silent mutationsSE 08405 24 D87Y Silent mutations None Silent mutationsSE 00103 16 D87Y Silent mutations None Silent mutationsSE 06505 16 D87Y Silent mutations None Silent mutationsSTM 03204 gt256 D87G None None NoneSE 08906 gt256 D87G Silent mutations None Silent mutationsSE 09506 gt256 D87G Silent mutations None Silent mutationsSE 07005 128 D87G Silent mutations None Silent mutationsSE 05004 64 D87G Silent mutations None Silent mutationsSE 01203 gt256 D87N Silent mutations None Silent mutationsSE 01503 gt256 D87N Silent mutations None Silent mutations


Table 1 Continued


SE 01303 96 D87N Silent mutations None Silent mutationsSTM 04305 gt256 S83F Silent mutations Silent mutations Silent mutationsSE 05505 gt256 S83F Silent mutations None Silent mutationsSE 05104 gt256 S83I Silent mutations None Silent mutationsSE 05204 gt256 S83I Silent mutations None Silent mutationsSE 09906 gt256 S83Y Silent mutations None Silent mutationsSTM 01803 24 None Silent mutations None M438ISTM 07107 64 None Silent mutations None NoneSTM 05505 32 None Silent mutations None NoneSTM 00602 16 None Silent mutations None NoneSTM 03304 16 None Silent mutations None NoneSTM 05305 16 None Silent mutations None NoneSTM 05605 16 None Silent mutations None NoneSTM 03404 12 None Silent mutations None NoneSTM 04805 8 None Silent mutations None NoneSTM 05705 6 None None None NoneD aspartic acid F phenylalanine G glycine I isoleucineMmethionine N asparagine S serine V valine Y tyrosine ldquosilentmutationsrdquo denotes synonymousbase substitutions in the QRDR of the target genes and thus does not result in amino acid change ldquononerdquo denotes wild-type sequence in the QRDR that isbase substitution did not take placeaThe first letter indicates the original amino acid followed by numbers that indicate the position of the amino acid in respective gene and the last letter indicatesthe substituting amino acid For example D87Y means tyrosine replaced aspartic acid in position 87 in GyrA subunit

from 60∘C (1min) to 95∘C (30 sec) with a ramp rate of 1 and60∘C for 15 sec (according to manufacturerrsquos instructions)The amplification plot for each sample was generated bythe 7500 software v20 (Applied Biosystems) linked to theinstrument The data generated was subsequently importedto the HRM software v201 (Applied Biosystems) for meltingcurve analysis The fluorescence change of each sample wasplotted against temperature and was normalized to producethe aligned melt curves In order to better illustrate theminor fluorescence changes between the wild type andmutants a difference plot was generated using the HRMsoftware In a difference plot one Salmonella strainwithwild-type sequence confirmed via sequencing was chosen as areference The fluorescence difference between each sampleand the reference was plotted against temperature Mutationsin the target region would produce a deviated curve in thedifference plot

3 Results

31 Detection of QRDR Mutations by Sequencing The gyrAgyrB parC and parE genes of 62 Salmonella strains weresubjected to sequencing Of these 62 strains missense muta-tions were detected in QRDR of gyrA (119899 = 52) and parE(119899 = 1) Silent mutations within and beyond QRDR wereobserved in gyrB (119899 = 60) parC (119899 = 1) and parE(119899 = 50) In QRDR of gyrA missense mutations occurredonly in codons 83 (conversion of serine to phenylalanine(S83F) tyrosine (S83Y) and isoleucine (S83I)) and 87 (con-version of aspartic acid to glycine (D87G) tyrosine (D87Y)

and asparagine (D87N)) In the parE gene two missensemutations were detected one within the QRDR (methionineto isoleucine (M438I)) and another outside of the QRDR(valine to phenylalanine (V521F)) (Table 1) The MIC datafor all 62 strains are shown in Table 1 In general mutationsat the QRDR of gyrA correlate with increased resistanceto nalidixic acid (MIC 16ndashgt256120583gmL) In the absence ofmutations (STM05705) the strain showed lowest MIC value(MIC 6120583gmL) Detailed genetic analyses of the QRDRs ofeach Salmonella strain are listed in the Supplementary Table(Table S1)

32 HRM Analysis for Rapid Mutation Screening of Gyraseand Topoisomerase IV Genes HRM primers were designedto span the QRDRs of gyrA (Ala67-Gln106) gyrB (Asp426-Lys447) parC (Ala64-Gln103) and parE (Asp420-Lys441)genes The primer pairs produce amplicons ranging from150 to 250 bp (Table 2) with a melting temperature ofapproximately 60∘C HRM analysis showed distinct meltcurves for wild-type versus mutant strains which wereconfirmed by sequencing Using sequencing as a guidelinetwo QRDR mutation-free strains were selected as referencesto generate difference plots (STM00602 for gyrA parC andparE genes STM03204 for gyrB gene) The QRDRs of gyrAparC and parE genes in the reference strain STM00602and the gyrB QRDR in the reference strain STM03204have matching sequences with the Salmonella Typhimuriumreference genomeLT2 (NC 003197)Detailed information forthese two reference strains is listed in the SupplementaryTable (Table S1)


Table 2 PCR primer sequences for HRM analysis of gyrase andtopoisomerase IV genes

Primer Sequence (51015840-31015840)b Amplicon size (bp)gyrA-F CAATGACTGGAACAAAGCCTA 164gyrA-R AACCGAAGTTACCCTGACCAgyrB-F TGTCCGAACTGTACCTGGTG 198gyrB-R ACTCGTCGCGACCGATACparC-F CGTCTATGCGATGTCAGAGC 219parC-R ATCGCCGCGAATGACTTCparE-F TACCGCGCAGGATCTTAATC 193parE-R GATCGCCACGGAAATATCATbPrimers designed in this study

The melting temperature of wild-type gyrA allele is 825ndash828∘C while that of the mutants (D87Y D87N S83F andS83Y) is at 820ndash824∘C Interestingly Salmonella strains withgyrAD87Gmutation produced a distinct melting curve com-pared to othermutants at slightly highermelting temperature(829ndash835∘C) The differences in the melting curves of thewild type versusmutants were distinguishable in both alignedand difference plot (Figure 1) The melting temperature forgyrB wild-type allele is 861∘Cndash862∘C while that of themutants is 862ndash867∘C Onemutant (STM04305) with threemutations in gyrB has a slightly lower melting temperature at860∘C The aligned plot did not show any difference whencomparing the melt curves of wild-type versus mutant gyrBalleles However they can be distinguished in difference plot(Figure 2) For parC gene the melting temperature for wild-type allele is at 861ndash865∘C (98 melted below 864∘C) andthe mutant at 864∘C Although the differences in the meltingtemperature were small the mutant produced a uniquemelting curve in the difference plot when compared to thewild type (Figure 3) For parE gene the melting temperaturefor wild-type allele is at 846ndash849∘C whereas that of themutants containing multiple mutations is at 849ndash852∘COne mutant (STM01803) which harbors a single mutationin parE QRDR has a lower melting temperature at 839∘CThe differences in wild-type versus mutant melt curves wereapparent in both aligned and difference plot (Figure 4)

4 Discussion

The HRM primers designed in this study spanned the entireQRDR of all four target genes The QRDRs are short regionsof DNA making them suitable targets for HRM analysis(optimal amplicon length 150ndash350 bp)The transition of meltcurves in HRM analysis of all target regions successfullydiscriminated the wild types from mutants Although themelting transitions were not very distinct in aligned meltcurves especially for gyrB the melting transitions of themutants became apparent when displayed in a difference plotThese transitions are direct consequences of the mutationsin the target gene QRDRs since they are the only geneticvariation within the PCR region

Point mutations were identified in QRDRs of gyrAgyrB parC and parE Consistent with the literature [9 14]

our study identified that mutations in gyrA QRDR mostcommonly take place at codons 83 and 87 often resulting inamino acid change and are associated with the nalidixic acidresistance in Salmonella strains Gyrase A-DNA complex isthe primary target for quinolones in Salmonella [7] Henceprolonged usage of quinolones often selects for developmentof resistant strains with mutations in gyrA since suchmutations confer better resistance to quinolone comparedto that of gyrB mutants [9] HRM analysis of the gyrAsuccessfully resolved the mutant strains into three distinctmelting profiles The melting profiles of the D87G mutantswere highly divergent from those of the other mutants TheD87G variants consisted of a single base substitution fromadenosine to guanineThis type of mutation represents class ISNP which often results in a highermelting temperature shiftcompared to other classes of SNP

Mutation was not common in the QRDR of gyrB amongSalmonella strains in this study Furthermore most studiesdid not identify mutations in gyrB of quinolone-resistantSalmonella [9 14 28] The HRM primers designed in thisstudy covered a region larger than the QRDR with anadditional 132 bp Hence the HRM variants detected in thisstudy consisted of three point mutations outside of QRDRregions However all nucleotide changes resulted in silentmutations One strain (STM04305) consisted of a silentmutation in the QRDR and an additional three mutationscompared to other mutants This strain consisted of class ISNPs at two sites thus producing a melting profile differentfrom other variants The different variants can be resolved byHRM as distinct difference plots

Topoisomerase is the secondary target of quinolones inGram-negative bacteria Mutations within QRDRs of parCand parE genes are uncommon among Salmonella [9 1428] In this study amino acid changes were not detected inparC genes and were identified in parE gene of only twoSalmonella strains The strains used in this study showedclassical phenotype that is resistant to nalidixic acid butnot to ciprofloxacin These observations are in agreementwith previous reports which suggested that the mutations ofthese genes are most probably infrequently detected becausethey either are not important in quinolone-resistance or areonly required for higher level of resistance that is resistanceto fluoroquinolones [9] Multiple mutations were detectedboth within and beyond the QRDRs of parC and parE genesHowever these were mostly silent mutations HRM assaysuccessfully differentiated the mutants from the wild typeThe melting profiles of the mutants were highly divergentfrom those of the wild type as clearly shown in both alignedmelt curves and difference plots

Despite the high efficiency of HRM analysis in mutationscreening this assay could only detect the presence of basesubstitutionsThemelting profiles should not be used directlyfor variants classification without first validating with DNAsequencing False positive may occur due to synonymousmutations while false negative occurs due to mutationsoutside targeted regions or resistance caused by other mech-anisms To date sequencing remains the ultimate proof formutations that lead to amino acid changes and subsequently


Alig

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nce (

)

105

95

85

75

65

55

45

35

25

15

5

minus5

Temperature (∘C)

7875

7925

7975

8025

8075

8125

8175

8225

8275

8325

8375

8425

8475

8525

8575

8625

8675

8725

(a)

Temperature (∘C)

7875

7925

7975

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8175

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8275

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8675

8725

275

225

175

125

75

25

minus25

minus75

minus125

minus175

minus225

minus275

Diff

eren

ce

(b)

Figure 1 Representative HRM aligned melting curves (a) and difference plot (b) for mutations in gyrA QRDR The aligned melting curveswere set at 100 at the beginning and 0 at the end of melting process In the difference plot the melting curves represent the temperatureat which the amplicons were completely denatured The difference of the melting temperature among the samples is clearly illustrated in thedifference plot The reference strain is indicated by the horizontal black line in the difference plot and the wild-type samples are indicated asgreen line The blue curves derived frommutants with the missense mutation D87G whereas the red curves belong to other mutants (D87YD87N S83F and S83Y) Mutants with missense mutations D87Y D87N S83F and S83Y were denatured at similar melting temperature andtherefore form a tight cluster

0

100

90

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30

20

10Alig

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)

Temperature (∘C)

8200

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8300

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(a)

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ce

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75

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minus125

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8250

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8350

8400

8450

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8550

8600

8650

8700

8750

8800

8850

8900

8950

9000

(b)

Figure 2 RepresentativeHRMalignedmelting curves (a) anddifference plot (b) formutations in gyrBQRDRThe reference strain is indicatedby the horizontal black line in the difference plot while mutants are represented by red (6 mutations within the HRM target region) or bluecurves (3 mutations within the HRM target region) All gyrBmutants are silent mutations

0

100

90

80

70

60

50

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30

20

10Alig

ned

fluor

esce

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)

Temperature (∘C)

8200

8250

8150

8300

8350

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8450

8500

8550

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8650

8700

8750

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(a)

9

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1

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eren

ce

minus1

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8200

8250

8150

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

(b)

Figure 3 Representative HRM aligned melting curves (a) and difference plot (b) for mutations in parC QRDR The reference strain isindicated by the horizontal black line in difference plot Wild-type samples are indicated by green curves and the single mutant is indicatedby red curve


5

105

95

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55

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fluor

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nce (

)

minus5

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790

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850

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870

880

890

900

910

(a)

4

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0

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eren

ce

minus2

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minus6

minus8

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(b)

Figure 4 Representative HRM aligned melting curves (a) and difference plot (b) for mutations in parE QRDR The reference strain isindicated by the horizontal black line in difference plot Wild-type samples are indicated by green curves and mutants are indicated by redcurves All mutant strains contain similar nucleotide changes in the QRDR resulting in tightly clustered melting curves

an alteration of enzyme structure [29] Nevertheless melt-ing profiles are useful parameters to distinguish mutantswith different characteristic and number of mutations sinceindividual mutant presents a distinctive melt curve whenviewed in the difference plot Hence HRM analysis can beconsidered as an efficient and cost-effective preliminary stepin the process of identifying QRDR mutations in Salmonella

In conclusion HRM analysis allows for detection ofmutations in Salmonella gyrase and topoisomerase IV geneswith sufficient sensitivity The adoption of this assay formutation screening prior to DNA sequencing is an attractiveoption to reduce the cost for mutational studies related toquinolone resistance Furthermore HRM analysis is time-saving allows for high throughput screening and is easy toperform since there is no post-PCRprocessing or purificationsteps Nevertheless sequencing shall be used for final valida-tion and should not be completely replaced by HRM analysis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge University of Malaya for the facil-ities and financial support The study was financially sup-ported by Ministry of Science Technology and Innovation(MOSTI) Science Fund (GA013-2013) High Impact Research(HIR) Grant (UMC625HIRMOHE02) and Post Gradu-ate Research Fund (PPP) (PS3192010B) They thank K HChua and C S J Teh for advice in the initial setup of HRMSoo Tein Ngoi was a recipient of the UM fellowship

References

[1] B Coburn G A Grassl and B B Finlay ldquoSalmonella the hostand disease a brief reviewrdquo Immunology and Cell Biology vol85 no 2 pp 112ndash118 2007

[2] M L Ling and G C Y Wang ldquoEpidemiological analysis ofSalmonella enteritidis isolates in Singaporerdquo Journal of Infectionvol 43 no 3 pp 169ndash172 2001

[3] P Padungtod and J B Kaneene ldquoSalmonella in food animalsand humans in northern Thailandrdquo International Journal ofFood Microbiology vol 108 no 3 pp 346ndash354 2006

[4] D Benacer K L Thong H Watanabe and S D Puthuc-heary ldquoCharacterization of drug-resistant Salmonella entericaserotype typhimurium by antibiograms plasmids integronsresistance genes and PFGErdquo Journal of Microbiology andBiotechnology vol 20 no 6 pp 1042ndash1052 2010

[5] K L Thong and S Modarressi ldquoAntimicrobial resistant genesassociated with Salmonella from retail meats and street foodsrdquoFood Research International vol 44 no 9 pp 2641ndash2646 2011

[6] J Ruiz ldquoMechanisms of resistance to quinolones target alter-ations decreased accumulation and DNA gyrase protectionrdquoJournal of Antimicrobial Chemotherapy vol 51 no 5 pp 1109ndash1117 2003

[7] G B Michael P Butaye A Cloeckaert and S SchwarzldquoGenes and mutations conferring antimicrobial resistance inSalmonella an updaterdquoMicrobes and Infection vol 8 no 7 pp1898ndash1914 2006

[8] D J Eaves L Randall D T Gray et al ldquoPrevalence ofmutationswithin the quinolone resistance-determining region of gyrAgyrB parC and parE and association with antibiotic resis-tance in quinolone-resistant Salmonella entericardquoAntimicrobialAgents and Chemotherapy vol 48 no 10 pp 4012ndash4015 2004

[9] K L Hopkins R H Davies and E J Threlfall ldquoMechanisms ofquinolone resistance in Escherichia coli and Salmonella recentdevelopmentsrdquo International Journal of Antimicrobial Agentsvol 25 no 5 pp 358ndash373 2005

[10] A Preisler M A Mraheil and P Heisig ldquoRole of novel gyrAmutations in the suppression of the fluoroquinolone resistancegenotype of vaccine strain SalmonellaTyphimuriumvacT (gyrAD87G)rdquo Journal of Antimicrobial Chemotherapy vol 57 no 3pp 430ndash436 2006

[11] F-X Weill S Bertrand F Guesnier S Baucheron P A D Gri-mont and A Cloeckaert ldquoCiprofloxacin-resistant SalmonellaKentucky in travelersrdquo Emerging Infectious Diseases vol 12 no10 pp 1611ndash1612 2006

[12] S Chen S Cui P F McDermott et al ldquoContribution oftarget gene mutations and efflux to decreased susceptibility of


Salmonella enterica serovar typhimurium to fluoroquinolonesand other antimicrobialsrdquoAntimicrobial Agents andChemother-apy vol 51 no 2 pp 535ndash542 2007

[13] C Kehrenberg A de Jong S Friederichs A Cloeckaert andS Schwarz ldquoMolecular mechanisms of decreased susceptibilityto fluoroquinolones in avian Salmonella serovars and theirmutants selected during the determination of mutant preven-tion concentrationsrdquo Journal of Antimicrobial Chemotherapyvol 59 no 5 pp 886ndash892 2007

[14] A D Lunn A Fabrega J Sanchez-Cespedes and J VilaldquoPrevalence ofmechanisms decreasing quinolone-susceptibilityamong Salmonella spp clinical isolatesrdquo International Microbi-ology vol 13 no 1 pp 15ndash20 2010

[15] C T Wittwer G H Reed C N Gundry J G Vandersteen andR J Pryor ldquoHigh-resolution genotyping by amplicon meltinganalysis using LCGreenrdquo Clinical Chemistry vol 49 no 6 pp853ndash860 2003

[16] M Liew R Pryor R Palais et al ldquoGenotyping of single-nucleotide polymorphisms by high-resolution melting of smallampliconsrdquo Clinical Chemistry vol 50 no 7 pp 1156ndash11642004

[17] A T Pietzka A Stoger S Huhulescu F Allerberger and WRuppitsch ldquoGene scanning of an internalin B gene fragmentusing high-resolution melting curve analysis as a tool for rapidtyping of Listeria monocytogenesrdquo The Journal of MolecularDiagnostics vol 13 no 1 pp 57ndash63 2011

[18] G H Reed and C T Wittwer ldquoSensitivity and specificity ofsingle-nucleotide polymorphism scanning by high-resolutionmelting analysisrdquo Clinical Chemistry vol 50 no 10 pp 1748ndash1754 2004

[19] H White and G Potts Mutation Scanning by High ResolutionMelt Analysis Evaluation of Rotor-Gene 6000 (Corbett LifeScience) HR-1 and 384-Well Lightscanner (Idaho Technology)National Genetics Reference Laboratory Wessex UK 2006

[20] R Slinger D Bellfoy M Desjardins and F Chan ldquoHigh-resolution melting assay for the detection of gyrA mutationscausing quinolone resistance in Salmonella enterica serovarsTyphi and Paratyphirdquo Diagnostic Microbiology and InfectiousDisease vol 57 no 4 pp 455ndash458 2007

[21] B M Loveless A Yermakova D R Christensen et al ldquoIden-tification of ciprofloxacin resistance by SimpleProbe HighResolution Melt and Pyrosequencing nucleic acid analysis inbiothreat agents Bacillus anthracis Yersinia pestis and Fran-cisella tularensisrdquo Molecular and Cellular Probes vol 24 no 3pp 154ndash160 2010

[22] X Chen F Kong Q Wang C Li J Zhang and G L GilbertldquoRapid detection of isoniazid rifampin and ofloxacin resistancein Mycobacterium tuberculosis clinical isolates using high-resolution melting analysisrdquo Journal of Clinical Microbiologyvol 49 no 10 pp 3450ndash3457 2011

[23] A S G Lee D C T Ong J C L Wong G K H Siu andW-C Yam ldquoHigh-resolution melting analysis for the rapiddetection of fluoroquinolone and streptomycin resistance inMycobacterium tuberculosisrdquo PLoS ONE vol 7 no 2 Article IDe31934 2012

[24] S T Ngoi B-A Lindstedt H Watanabe and K L ThongldquoMolecular characterization of Salmonella enterica serovarTyphimurium isolated from human food and animal sourcesin Malaysiardquo Japanese Journal of Infectious Diseases vol 66 no3 pp 180ndash188 2013

[25] S T Ngoi and K LThong ldquoMolecular characterization showedlimited genetic diversity among Salmonella Enteritidis isolated

fromhumans and animals inMalaysiardquoDiagnosticMicrobiologyand Infectious Disease vol 77 no 4 pp 304ndash311 2013

[26] A Fabrega L du Merle C Le Bouguenec M T J de Anta andJ Vila ldquoRepression of invasion genes and decreased invasion ina high-level fluoroquinolone-resistant Salmonella typhimuriummutantrdquo PLoS ONE vol 4 no 11 Article ID e8029 2009

[27] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[28] K-Y Kim J-H Park H-S Kwak and G-J Woo ldquoCharacter-ization of the quinolone resistance mechanism in foodborneSalmonella isolates with high nalidixic acid resistancerdquo Interna-tional Journal of Food Microbiology vol 146 no 1 pp 52ndash562011

[29] E A Tindall D C Petersen P Woodbridge K Schipany andV M Hayes ldquoAssessing high-resolution melt curve analysis foraccurate detection of gene variants in complexDNA fragmentsrdquoHuman Mutation vol 30 no 6 pp 876ndash883 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


linked to point mutations in QRDR of the gyrase genes[13 14] While most studies revealed that codon 87 of gyrAgene represents the most frequently mutated amino acid inSalmonella [8 12 14] point mutations outside of QRDR mayalso play a role in quinolone-resistance [6] NeverthelessQRDR remains the major focus in quinolone-resistancestudies since this region exhibits high mutation rate

DNA sequencing is the gold standard for detectinggenetic changes However it is relatively costly to sequenceall four genes when examining a large sample pool Fur-thermore mutations are rare in parC and parE genes [9]therefore DNA sequencing will not be an economicallyviable option when dealing with such conserved regionsThereal-time polymerase chain reaction- (PCR-) based high-resolution melt curve (HRM) analysis is able to detect smallgenetic variation in PCR amplicons [15ndash17] This methodis highly sensitive and specific and has been employed insingle nucleotide polymorphism (SNP) scanning [18]DuringHRM analysis a DNA intercalating dye is included in thePCR reaction mixture This compound interacts specificallywith double-stranded DNA and emits fluorescence signalbut loses fluorescence when released from the DNA duringdenaturationUpon completion of PCR theDNAsamples aresubject to a temperature gradient and the loss of fluorescencesignal resulting from denaturation gives each DNA samplea unique melting curve that is detected by the real-timePCR system [19] Hence different Salmonella strains can bediscriminated based on their sequence length GC contentor strand composition

HRM assay has been used for the identification ofquinolone-resistance strains in pathogens such as Mycobac-terium tuberculosis Bacillus anthracis Yersinia pestis Fran-cisella tularensis and Salmonella Typhi and Paratyphi A [20ndash23] In these studies only gyrA gene was examined Althoughinfrequent mutations in gyrB parC and parE genes ofSalmonella and their correlation with lower susceptibility toquinolones and fluoroquinolones have been documented too[9] The objective of the study was to determine the potentialof HRM analysis for rapid screening of mutations in QRDRof Salmonella gyrase and topoisomerase IV genes

2 Materials and Methods

21 Bacterial Strains A total of 195 Salmonella enterica strainswere obtained from the culture collection of Laboratory ofBiomedical Sciences and Molecular Microbiology Instituteof Graduate Studies University of Malaya The susceptibilityof the strains towards nalidixic acid and ciprofloxacin wasexamined and reported in previous studies [24 25] Sixty-two Salmonella strains showing resistance or reduced sus-ceptibility towards nalidixic acid were used to develop theHRM assay in this study (Salmonella Typhimurium 119899 =12 Salmonella Enteritidis 119899 = 50) All selected strainswere susceptible to ciprofloxacin The minimum inhibitoryconcentration (MIC) of nalidixic acid for the strains wasdetermined by using Etest strips (BioMerieux Marcy lrsquoEtoileFrance) The test was performed according to manufacturerrsquosinstructions Genomic DNA for each bacterial strain was

extracted by using Wizard genomic DNA purification kit(Promega Madison USA) The details of all Salmonellastrains used in this study are listed in the SupplementaryTable (see Table S1 in the Supplementary Material availableonline at httpdxdoiorg1011552014718084)

22 DNA Sequencing PCR amplification of gyrA gyrBparC and parE QRDRs was performed using previouslydescribed primers [26] The selected primers amplify theQRDRs of the respective genes PCR amplification wasdone in a 25 120583L monoplex reaction mixture containing1x colourless GoTaq Flexi Buffer 15mmolL magnesiumchloride (MgCl

2) 200120583molL deoxynucleoside triphosphate

(dNTP) mix 03 120583molL of each primer pair 1 U Taq DNApolymerase (Promega Madison USA) and approximately100 ng of bacterial genomicDNAThe PCR reactionmixtureswere first incubated at 94∘C for 2min followed by 25 cycles of94∘C for 30 s 60ndash63∘C (annealing temperature varies for eachgene) for 30 s and 72∘C for 45 s with a final extension step of72∘C for 5min The appropriate annealing temperatures forgyrA gyrB parC and parE are 60∘C 60∘C 63∘C and 62∘Crespectively Agarose gel electrophoresis was then carriedout to confirm the presence of amplicons prior to DNAsequencing Next the PCR products were purified usingWizard SV gel and PCR clean-up system (PromegaMadisonUSA) and the purified PCR products were sent to First BASELaboratories (Selangor Malaysia) for sequencing Mutationsin the target genes were determined by comparison withthe wild-type sequence of the respective genes in SalmonellaTyphimuriumLT2 (GenBank accession numberNC 003197)

23 HRMAnalysis A homology search for gyrA gyrB parCand parE gene sequences of Salmonella serovars from theNCBI GenBank database was performed Sequence align-ment was done using the molecular evolutionary geneticsanalysis software version 5 (MEGA5) [27] Primers weredesigned using the online tool Primer3Plus (httpprimer3-pluscomcgi-bindevprimer3pluscgi) spanning the QRDRof each gene of interest and were commercially synthe-sized (NHK Bioscience Solutions Kuala Lumpur Malaysia)Approximately 20 ng of bacterial genomic DNA was addedinto 1x MeltDoctor HRM master mix (Applied BiosystemsFoster City CA USA) which was premixed with 03120583Mof each primer pair Sterile deionized distilled water wasadded to make up a final reaction volume of 20 120583L Thereproducibility of the assay was determined by performingall reactions in triplicate In each assay the DNA samplefrom awild-type reference strain was used as positive controlThe selection of the wild-type reference strains for thedifferent target genes was explained in the Results section(Section 32) Negative control for the experiment was theHRM reaction mix without the addition of bacterial DNAThe HRM assay was performed on the 7500 Fast Real-Time PCR System (Applied Biosystems) The real-time PCRamplification included a holding stage at 95∘C for 10minfollowed by the cycling stage with 40 cycles of 95∘C for 15 secand subsequently 60∘C for 1min The subsequent melt curvestage consisted of the following steps 95∘C for 15 sec a melt






Table 1 Continued




3 Results








4 Discussion








Alig

ned

fluor

esce

nce (

)

105

95

85

75

65

55

45

35

25

15

5

minus5

Temperature (∘C)

7875

7925

7975

8025

8075

8125

8175

8225

8275

8325

8375

8425

8475

8525

8575

8625

8675

8725

(a)

Temperature (∘C)

7875

7925

7975

8025

8075

8125

8175

8225

8275

8325

8375

8425

8475

8525

8575

8625

8675

8725

275

225

175

125

75

25

minus25

minus75

minus125

minus175

minus225

minus275

Diff

eren

ce

(b)


0

100

90

80

70

60

50

40

30

20

10Alig

ned

fluor

esce

nce (

)

Temperature (∘C)

8200

8250

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

8950

9000

(a)

Diff

eren

ce

125

75

25

minus125

minus75

minus25

Temperature (∘C)

8200

8250

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

8950

9000

(b)


0

100

90

80

70

60

50

40

30

20

10Alig

ned

fluor

esce

nce (

)

Temperature (∘C)

8200

8250

8150

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

(a)

9

7

5

3

1

Diff

eren

ce

minus1

minus3

minus5

Temperature (∘C)

8200

8250

8150

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

(b)



5

105

95

85

75

65

55

45

35

25

15

Alig

ned

fluor

esce

nce (

)

minus5

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(a)

4

2

0

Diff

eren

ce

minus2

minus4

minus6

minus8

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(b)






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Table 1 Continued




3 Results








4 Discussion








Alig

ned

fluor

esce

nce (

)

105

95

85

75

65

55

45

35

25

15

5

minus5

Temperature (∘C)

7875

7925

7975

8025

8075

8125

8175

8225

8275

8325

8375

8425

8475

8525

8575

8625

8675

8725

(a)

Temperature (∘C)

7875

7925

7975

8025

8075

8125

8175

8225

8275

8325

8375

8425

8475

8525

8575

8625

8675

8725

275

225

175

125

75

25

minus25

minus75

minus125

minus175

minus225

minus275

Diff

eren

ce

(b)


0

100

90

80

70

60

50

40

30

20

10Alig

ned

fluor

esce

nce (

)

Temperature (∘C)

8200

8250

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

8950

9000

(a)

Diff

eren

ce

125

75

25

minus125

minus75

minus25

Temperature (∘C)

8200

8250

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

8950

9000

(b)


0

100

90

80

70

60

50

40

30

20

10Alig

ned

fluor

esce

nce (

)

Temperature (∘C)

8200

8250

8150

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

(a)

9

7

5

3

1

Diff

eren

ce

minus1

minus3

minus5

Temperature (∘C)

8200

8250

8150

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

(b)



5

105

95

85

75

65

55

45

35

25

15

Alig

ned

fluor

esce

nce (

)

minus5

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(a)

4

2

0

Diff

eren

ce

minus2

minus4

minus6

minus8

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(b)






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





4 Discussion








Alig

ned

fluor

esce

nce (

)

105

95

85

75

65

55

45

35

25

15

5

minus5

Temperature (∘C)

7875

7925

7975

8025

8075

8125

8175

8225

8275

8325

8375

8425

8475

8525

8575

8625

8675

8725

(a)

Temperature (∘C)

7875

7925

7975

8025

8075

8125

8175

8225

8275

8325

8375

8425

8475

8525

8575

8625

8675

8725

275

225

175

125

75

25

minus25

minus75

minus125

minus175

minus225

minus275

Diff

eren

ce

(b)


0

100

90

80

70

60

50

40

30

20

10Alig

ned

fluor

esce

nce (

)

Temperature (∘C)

8200

8250

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

8950

9000

(a)

Diff

eren

ce

125

75

25

minus125

minus75

minus25

Temperature (∘C)

8200

8250

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

8950

9000

(b)


0

100

90

80

70

60

50

40

30

20

10Alig

ned

fluor

esce

nce (

)

Temperature (∘C)

8200

8250

8150

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

(a)

9

7

5

3

1

Diff

eren

ce

minus1

minus3

minus5

Temperature (∘C)

8200

8250

8150

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

(b)



5

105

95

85

75

65

55

45

35

25

15

Alig

ned

fluor

esce

nce (

)

minus5

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(a)

4

2

0

Diff

eren

ce

minus2

minus4

minus6

minus8

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(b)






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Alig

ned

fluor

esce

nce (

)

105

95

85

75

65

55

45

35

25

15

5

minus5

Temperature (∘C)

7875

7925

7975

8025

8075

8125

8175

8225

8275

8325

8375

8425

8475

8525

8575

8625

8675

8725

(a)

Temperature (∘C)

7875

7925

7975

8025

8075

8125

8175

8225

8275

8325

8375

8425

8475

8525

8575

8625

8675

8725

275

225

175

125

75

25

minus25

minus75

minus125

minus175

minus225

minus275

Diff

eren

ce

(b)


0

100

90

80

70

60

50

40

30

20

10Alig

ned

fluor

esce

nce (

)

Temperature (∘C)

8200

8250

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

8950

9000

(a)

Diff

eren

ce

125

75

25

minus125

minus75

minus25

Temperature (∘C)

8200

8250

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

8950

9000

(b)


0

100

90

80

70

60

50

40

30

20

10Alig

ned

fluor

esce

nce (

)

Temperature (∘C)

8200

8250

8150

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

(a)

9

7

5

3

1

Diff

eren

ce

minus1

minus3

minus5

Temperature (∘C)

8200

8250

8150

8300

8350

8400

8450

8500

8550

8600

8650

8700

8750

8800

8850

8900

(b)



5

105

95

85

75

65

55

45

35

25

15

Alig

ned

fluor

esce

nce (

)

minus5

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(a)

4

2

0

Diff

eren

ce

minus2

minus4

minus6

minus8

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(b)






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5

105

95

85

75

65

55

45

35

25

15

Alig

ned

fluor

esce

nce (

)

minus5

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(a)

4

2

0

Diff

eren

ce

minus2

minus4

minus6

minus8

Temperature (∘C)

790

800

810

820

830

840

850

860

870

880

890

900

910

(b)






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article high resolution melting analysis for rapid...

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