Research Article The Development of DNA Based Methods …downloads.hindawi.com/journals/ijac/2016/4352308.pdf · Factory sample I (tobacco stems, no sulphate cellulose); ... of the

Research ArticleThe Development of DNA Based Methods for theReliable and Efficient Identification of Nicotiana tabacum inTobacco and Its Derived Products

Sukumar Biswas1 Wei Fan1 Rong Li1 Sifan Li1 Wenli Ping2

Shujun Li2 Alexandra Naumova3 Tamara Peelen4 Esther Kok5

Zheng Yuan1 Dabing Zhang1 and Jianxin Shi1

1 Joint International Research Laboratory of Metabolic amp Developmental SciencesSJTU-University of Adelaide Joint Centre for Agriculture and Health School of Life Sciences and Biotechnology800 Dongchuan Road Minghan District Shanghai 200240 China2Institute of Tobacco Research Henan Academy of Agricultural Sciences Xuchang Henan 461000 China3Central Customs Laboratory Bulgarian Customs Agency G S Rakovski Street 47 1202 Sofia Bulgaria4Dutch Customs Laboratory Kingsfordweg 1 1043 GN Amsterdam Netherlands5RIKILT Wageningen University and Research Centre PO Box 230 6700 AE Wageningen Netherlands

Correspondence should be addressed to Jianxin Shi jianxinshisjtueducn

Received 21 April 2016 Accepted 26 July 2016

Academic Editor Gunther K Bonn

Copyright copy 2016 Sukumar Biswas et alThis is an open access article distributed under theCreativeCommonsAttributionLicensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smugglingand classify tariff and excise in tobacco industry to control illegal tobacco trade In this study two sensitive and specific DNA basedmethods one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay weredeveloped for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples andcommodities Both assays targeted the same sequence of the uridine 51015840-monophosphate synthase (UMPS) and their specificitiesand sensitivities were determined with various plant materials Both qPCR and LAMP methods were reliable and accurate in therapid detection of tobacco components in various practical samples including customs samples reconstituted tobacco samplesand locally purchased cigarettes showing high potential for their application in tobacco identification particularly in the specialcases where the morphology or chemical compositions of tobacco have been disruptedTherefore combining both methods wouldfacilitate not only the detection of tobacco smuggling control but also the detection of tariff classification and of excise

1 Introduction

Tobacco (Nicotiana tabacum L) is cultivated as an importantcommercial crop worldwide for the production of cigarettescigars and other tobacco products [1] In 2014 the globaltobacco industry sold about 56 trillion cigarettes and itsmar-ket value is around US$744 billion [2] Because of its massiveeconomic importance there is increasing illicit tobacco tradeincluding smuggling [3] Globally about 657 billion cigarettesvalued at around US$405 million were smuggled yearly[4] Illicit tobacco trade is the main hindrance to collecting

tobacco excises and taxes and once eliminated governmentrevenue would gain at least US$31 billion globally [5]

There are mainly three types of tobacco products onthe market cigarettes cigars and fine-cut tobacco (smokingtobacco) A cigarette is a roll of fine-cut tobacco having awrapper of thin paper A cigar consists of tobacco rolledin a binder of tobaccoreconstituted tobacco and an outerwrapper of tobacco The outer wrapper of a cigar can alsobe made of reconstituted tobacco for instance in cigarillosa short narrow cigar For tax and excise purposes these def-initions are used and classification of these tobacco products

Hindawi Publishing CorporationInternational Journal of Analytical ChemistryVolume 2016 Article ID 4352308 6 pageshttpdxdoiorg10115520164352308

2 International Journal of Analytical Chemistry

Table 1 Descriptions of plant samples used in this study

Plant materials Description of the plant materials Providers

Nontobacco plants(fresh leaves)

Alfalfa (Medicago sativa) Altingia (Altingia gracilipes) canola (Brassica campestris)castor (Ricinus communis) Chinese jasmine (Jasminum officinale) coriander(Coriandrum sativum) Daphniphyllum (Daphniphyllum teijsmannii) eggplant

(Solanum melongena) garden petunia (Petunia times hybrid) Indica rice (Oryza sativavar indica) mondo grass (Ophiopogon japonicus) Pittosporum (Pittosporum

tobira) pomegranate (Punica granatum) sapodilla (Manilkara zapota) spinach(Spinacia oleracea) watermelon (Citrullus lanatus)

Locally collected

Dried tobacco leaves Burley (3) Oriental SAADI-6 Virginia (2) Oriental (5) reconstituted tobaccofoiled (1) Virginia grade A (1) Virginia grade B (1) Oriental BASMAK Bulgarian customs

Fresh tobacco leaves Burley Maryland Locally collected

Reconstituted tobaccoFactory sample I (tobacco stems no sulphate cellulose) factory sample II (notobacco stems no sulphate cellulose) factory sample III (tobacco stems with

sulphate cellulose) commercial wrapperDutch customs

Tobacco stem Tobacco stems Bulgarian customsCigarettes Liqun Double Happiness Mevius Marlboro Wuyeshen Locally purchasedThe numbers in brackets indicate the numbers of tobacco varieties of this species tested in the experiment

is therefore mainly based on the composition of the outerwrapper

Reconstituted tobacco is made from a pulp of mashedtobacco waste [6 7] The definition of reconstituted tobacco(Harmonised System World Customs Organisation) is asfollows ldquoit is made by agglomerating finely divided tobaccofrom tobacco leaves tobacco refuse or dust whether or noton a backing (eg sheet of cellulose from tobacco stems rdquoProducts with a wrapper of reconstituted tobacco whichmay consist partly of substances other than tobacco arealso seen as cigarscigarillos Because the differences ofexcise duties between cigarettes and cigars are significant inmany European countries [8] it is necessary to detect anyretail cigarscigarillos that do not match the definition of acigarcigarillo and should therefore be sold as a cigaretteTherefore a reliable method is needed to detect the presenceof tobacco in outer wrappers of these tobacco products

Tobacco identification is usually based on the detectionof the presence of nicotine neophytadiene [6] vitamin E[9] and microscopic fragments [10] Although these iden-tification methods are still used in certain cases (especiallyin reconstituted tobacco) they often failed to work [7] Inpractice microscopic tobacco fragments may not be detectedin reconstituted tobacco and on the other hand fraudulentactions may include the spraying or impregnation of nico-tine and neophytadiene into nontobacco constituents Thusalternative methods are required for successful detection andidentification of tobacco In EU customs officers use trainedanimals such as dogs [11ndash13] and giant African pouchedrats (Cricetomys gambianus) [13] to successfully detect thesmell of tobacco However because the odors of tobaccocan be readily enhanced or eliminated this method is likelyuseful to some extent for tobacco smuggling but obviouslynot applicable for tariff classification and for excise whichneedsDNAbasedmethods In a previous study a quantitativereal-time PCR (qPCR) assay based on a putrescine N-methyltransferase (PMT1) gene PMT1a was developed toidentifyNicotiana tabacum in tobacco products [7] However

because there is another PMT1b gene in tobacco genome andthe sequence of PMT1b was not available in the database [7]therefore the primers used for PMT1a are likely nonspecificto PMT1a In addition besides PMT1 3 additional PMT(PMT2ndashPMT4) genes with high similarity to each otherare present in tobacco genome [7] which makes the useof PMT1a as an endogenous reference gene of tobacco lessreliable

In this study by targeting a putative single copy tobaccoendogenous gene sequence of the uridine 51015840-monophosphatesynthase (UMPS) we developed two DNA based methodsa novel quantitative real-time PCR (qPCR) method and aloop-mediated isothermal amplification (LAMP) methodto detect the presence of tobacco components in variouscommodities The specificity and sensitivity were assayedusing various available materials including customs samplesreconstituted tobacco samples cured tobacco samples andlocally purchased cigarettes Our results showed that thenovel qPCR based on UMPS had 15-fold lower limit ofdetection and better specificity than the previously reportedPMT1a based qPCR method [7] Both qPCR and LAMPdeveloped on UMPS could be useful for rapid tobaccoidentification in a reliable and efficient way

2 Materials and Methods

21 PlantMaterials All the plantmaterials used in this studyincluding different tobacco and nontobacco materials andproducts are listed in Table 1

22 Genomic DNA Extraction and Purification A commer-cial DNA extraction kit the DNeasy Plant Mini kit (QiagenShanghai China) was used to extract genomic DNA 20mglyophilized tissue of different plant materials including freshtobacco leaves unprocessed tobacco reconstituted tobaccotobacco stems cigarettes and cigarette white wrapperswas considered for DNA isolation Briefly the sample was

International Journal of Analytical Chemistry 3

collected and washed with 1x PBS buffer before starting DNAextraction Then the sample was ground in the presence ofliquid nitrogen DNA was extracted following the instruc-tions given by the DNeasy Plant Mini kit The qualities andquantities of extracted genomicDNA samples weremeasuredand evaluated using the NanoDrop 1000UVvis Spectropho-tometer (NanoDrop Technologies Inc Wilmington DEUSA) by observing OD260OD280 and OD260OD230 and1 (wv) agarose gel electrophoresis in 05x TBE with GelRedstaining All purifiedDNA samples were stored atminus20∘CuntilPCR analysis

23 DNA Oligonucleotide Primers and Probes All primersfor the LAMP assay were designed with the website-based software Primer ExplorerV4 (httpprimerexplorerjpelamp400indexhtml) and synthesized by Invitrogen CoLtd (Shanghai China) There are two outer and two innerprimers in the LAMP assay The two outer primers includeone forward outer primer (F3) and one backward outerprimer (B3) while the two inner primers include one forwardinner primer (FIP) and one backward inner primer (BIP)TheFIP consists of one F1c (complementary of F1) and one sensesequence F2 and the BIP consists of one B1c (complementaryof B1) and one sense sequence B2 [14] Meanwhile DNAoligonucleotide primers and TaqMan probes for qPCRwere designed with Primer Premier 50 and synthesized byInvitrogen Co Ltd (Shanghai China) The detailed primerand probe sequences used in this study are listed in Table S1 inSupplementary Material available online at httpdxdoiorg10115520164352308

24 LAMP Assay For the LAMP assay each reaction wasperformed in a 25120583L reaction mixture containing 20mMTris-HCl (pH 88) 10mM KCl 10mM (NH

4)2SO4 25mM

MgSO4 01 Triton X-100 5M betaine (Sigma USA) 10 120583M

of each FIP and BIP primer 10 120583M of each F3 and B3primer 2mM of each dNTP and 2120583L of purified genomicDNA template After the addition of DNA template thereaction mixture was incubated at 95∘C for 5min followedby immediate cooling down on ice and the addition of 8UBst DNA polymerase large fragment (New England Biolabs)The reaction mixture was then incubated at 64∘C for 60minin a TaKaRa PCR Thermal Cycler (TaKaRa Bio Inc Japan)Theno template control (NTC) containedwater instead of theDNA template and the LAMP assay was repeated three timesThe amplified LAMP products were examined either throughvisual inspection with 1000x SYBR Green I (Generay BiotechCo Ltd Shanghai China) or on agarose gel electrophoresis(AGE) analysis

25 qPCR Assay qPCR assays were performed on an ABI7900HT Fast Real-Time PCR System (Applied BiosystemsUSA) in 96-well plates A real-time PCR reaction mixture(25 120583L) contained the following reagents 125 120583L of TaqManGene Expression Master Mix (Applied Biosystems) 10 120583L(04 120583M) of each primer 05120583L (02 120583M) of the probe 50 120583Lof the ddH

2O and 50 120583L of the genomic DNA Real-time

PCR amplification procedures were carried out using the

following conditions one cycle of 10min at 95∘C followedby 45 cycles of 15 s at 95∘C and 60 s at 60∘C At the annealingstep the fluorescent signal was detected during every PCRcycle PCR data was analyzed using ABI 7900HT softwareSDS version 24 The real-time PCR reaction was repeated intriplicate with every three replicates

3 Results and Discussion

31 Genomic DNA Extraction Genomic DNA from freshleaves of different nontobacco and tobacco samples couldbe easily and rapidly extracted using DNeasy Plant Minikit (Qiagen Shanghai China) according to the instructionsgiven by the manufacturer and the obtained DNA wassuccessfully used directly for LAMP and qPCR assays inagreement with the previous study [7] However the genomicDNA isolated from driedcured tobacco leaves reconstitutedtobacco samples tobacco stems and cigarettes using the samekit was not pure enough for LAMP and qPCR assays There-fore an additional step of 2-3 times washing of those sampleswith 1x PBS buffer before DNA extraction was performedWe assumed that washing with PBS buffer would removecontaminants particularly dust inhibitoryinterfering chem-icals and microorganisms that break down DNA or interferewith subsequent DNA isolation and purity Indeed DNAsamples isolated following this strategy were found to havereliable and reproducible result in PCR amplification both inLAMP and in real-time PCR assays

32 Specificity Assay The tobacco gene UMPS is a bifunc-tional enzyme that catalyzes the last two steps of the con-served pyrimidine biosynthetic pathway in higher eukaryotesincluding tobacco In solanaceous species it appears in thegenome as only one copy [15] Therefore it was chosen inour study to investigate its suitability as a marker gene for thedetection of tobacco BLAST (Basic Local Alignment SearchTool) was then performed and a unique region for tobaccowas used for the design of the suitable primer sets for bothLAMP and qPCR assays (Table S1)

The specificity assay of UMPS in LAMP and qPCR wasperformed in one set of plant samples containing one tobaccopositive control (Table 1 and Table S2) As shown in Figure 1LAMP detected the typical ladder-like pattern product onlyin the positive control which was confirmed by the visualiza-tion test where the final green color also appeared in the posi-tive control (Figures 1(a) and 1(b)) qPCRassaywhich showedthe amplification of UMPS only in the positive control (Fig-ure 1(c)) also verified the specificity ofUMPSTherefore bothmethods that were developed on UMPS gene were highlyspecific to tobacco In this test we also included eggplantand petunia two plants within the same Solananceae familyas tobacco It is reported that eggplant produces nicotine aswell [16] The failure of the detection of UMPS in eggplantand petunia further indicated that both assays are specific totobacco which was also in agreement with our BLAST result

33 Sensitivity Assay The limit of detection (LOD) is thelowest amount or concentration of analyte that can be


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18M

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Sample ID

Ct

26252423

27

(c)

Figure 1 Specificity test of UMPS gene in tobacco and nontobaccoplants (a) LAMP method through direct visual detection withSYBR Green I (b) LAMPmethod on 2 agarose gel electrophoresisanalysis (c) qPCR method Lane 1 negative control (NTC) lane 2positive control (PTC) lanes 3ndash18 Chinese jasmine alfalfaAltingiacanola Pittosporum Daphniphyllum mondo grass sapodilla gar-den petunia castor oil indica rice coriander spinach pomegranatewatermelon and eggplant lane M Trans 2K DNA marker Ct wasexpressed as mean Ct plusmn SD from 3 independent experiments withthree replications

reliably detected through acceptable criterion [17] To deter-mine the LODs of the developed LAMP and qPCR assaysgenomic DNA isolated from fresh tobacco leaves unpro-cessed tobacco and reconstituted tobacco samples was seri-ally diluted to final concentrations of 60 30 15 75 375 188094 and 047 ng120583L for LAMP and 24 12 6 3 15 075 038019 0095 0048 0024 and 0012 ng120583L for qPCR by 1x TEbuffer respectively 2120583L and 5 120583L of each diluted DNA wereused as template in the LAMP assay and the qPCR assayrespectively

As shown in Figure 2 when fresh tobacco DNA was usedfor the LAMP assay the color change from orange to greenwas observed in all reactions of each diluted DNA level withthe exception of 094 ng120583L (Figure 2(a))This was confirmedby the agarose gel electrophoresis analysis (Figure 2(b))indicating that the absolute LOD of LAMP detection ofUMPS gene was 188 ng (094 ng120583L times 2120583L in a reaction)which was equivalent to about an average of 395 copies oftobacco genomic DNA (httpcelsuriedugsccndnahtml)The identical LAMP results of the sensitivity assay werealso obtained in assays with reconstituted tobacco samples(Figures S1A and 1B) and cured tobacco samples (Figures S2Aand 2B) In the qPCR assay standard curves were generatedby plotting the average Ct values versus known quantities

1 2 3 4 5 6 7 8 9

(a)

1 2 3 4 5 6 7 8 9M

(b)

DNA quantity (ng)

232425262728293031323334

Ct

Efficiency 92

12060301575375188094047

y = minus09118x + 33988

R2= 09981

012 024

(c)

Figure 2 Sensitivity test of UMPS gene using serial dilutions ofgenomic DNA from fresh tobaccoleave samples (a) LAMP methodthrough direct visual detection with SYBR Green I (b) LAMPmethod on 2 agarose gel electrophoresis analysis Lane 1 NTClanes 2ndash9 120 60 30 15 75 375 188 and 094 ng per reactionrespectively lane M Trans 2K DNA marker (c) qPCR method Forstandard curve a serial dilution of DNA samples (120 60 30 15 75375 188 094 047 024 012 and 006 ng) was usedThe result wasdeveloped after considering 3 independent experiments with threereplications

of genomic tobacco DNA (Figure 2(c)) and the LOD of theqPCR forUMPSwas 012 ng (0024 ng120583Ltimes 5 120583L in a reaction)when fresh tobacco samples were used which was equivalentto an average of 25 copies of tobacco genomicDNAand betterthan the LOD reported for PMT1a (037 ng equivalent to anaverage of 39 copies of tobacco genomic DNA) [7]The LODsof the qPCR for UMPS in reconstituted tobacco samples andcured tobacco samples were 047 ng and 024 ng respectivelyequivalent to about an average of 100 copies and 50 copies oftobacco genomic DNA respectively (Figures S1C and S2C)We repeated these experiments several times with seriallydiluted freshly isolated DNA and reproduced the results

Our abovementioned results indicated that although theextent of processing did not affect the sensitivity of LAMP


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19M

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Sample ID

2325272931333537

Ct

(c)

Figure 3 Detection of tobacco components in different tobac-cocultivars (a) LAMP method through direct visual detection withSYBR Green I (b) LAMPmethod on 2 agarose gel electrophoresisanalysis (c) qPCR method Lane 1 NTC lane 2 PTC lanes 3ndash1715 tobacco samples of different cured tobacco varieties lanes 18-19two fresh tobacco samples lane M Trans 2K DNA marker Ct wasexpressed as mean Ct plusmn SD from 3 independent experiments withthree replications

method it did significantly affect the sensitivity of the qPCRmethod It is reported that the DNA yield of processedtobacco samples differed significantly fromunprocessed sam-ples [7] Our study on the other hand indicated that evenif the same amount of DNA was used for the PCR reactionthe LODof qPCRwithDNA fromprocessed tobacco sampleswas higher than that with DNA from unprocessed tobaccosamples demonstrating that processing affected the integrityof the genomic DNA and caused the breakdown or damageto targeted genes In addition our results demonstrated thatthe qPCR method had about 4 to 10 times lower LOD thanthe LAMP method

34 Application in Various Practical Samples The devel-oped LAMP and real-time PCR methods were first usedto detect the presence of the targeted gene in differentcultivars of tobacco (listed in Table S2) As shown in Fig-ure 3 it seemed that the presence of UMPS was conservedin these tested tobacco cultivars and there is no singlenucleotide polymorphism in this region of the gene thatmay negatively affect its detection among these tobaccocultivars Thus the result strongly suggested that UMPScan serve as a tobacco specific gene for the detectionof tobacco components based on either LAMP or qPCRassay

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

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14M

(b)

23252729313335

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

CtSample ID

(c)

Figure 4 Practical sample detection with different practical sam-ples (a) LAMP method through direct visual detection with SYBRGreen I (b) LAMP method on 2 agarose gel electrophoresisanalysis (c) qPCR method Lane 1 NTC lane 2 PTC lanes 3-4 coriander and spinach lanes 5-6 burley and Oriental SAADI-6 lanes 7-8 NT Oriental and NT Virginia gold lanes 9-10reconstituted tobacco (no tobacco stems no sulphate cellulose) andreconstituted tobacco (tobacco stems with sulphate cellulose) lanes11-12 cigarette (Liqun and Double Happiness) lanes 13-14 wrappersfrom Liqun and Marlboro respectively lane M Trans 2K DNAmarker Ct was expressed as mean Ct plusmn SD from 3 independentexperiments with three replications

The developed LAMP and real-time PCR methods werethen used to detect the presence of tobacco in variouspractical samples like a positive tobacco control a negativecontrol and other samples such as nontobacco samplesreconstituted tobacco samples tobacco stems cigarettes andcigarette white wrappers (Table 1 and Table S3) As shown inFigure 4 and Figures S3ndashS5 both LAMP and qPCR methodscould accurately detect the presence of tobacco in samplescontaining tobacco components such as positive controltobacco stem reconstituted tobacco and cigarette samplesexcept cigarette white wrappers These results showed thatboth methods were effective and reliable for the detection ofthe presence of tobacco components regardless of the degreeof processing indicating that both are of high potential to beused for reliable and accurate detection of tobacco in customsservice for tariff clarification and excise

4 Conclusions

In this study we have established two DNA based meth-ods LAMP and qPCR to detect the presence of tobaccocomponents The former can be used for rapid and visual


identification of tobacco components as LAMP methodusually does in other DNA based detection cases [18] whichis particularly useful for on-site assays while the later can beused for accurate qualitative identification of tobacco and itsproducts for routine analysis in the lab which has lower LODthan the previous TaqMan method [7] Both are applicablenot only to fresh samples but also to processed samples inwhich tobacco morphology or chemical composition hasbeen altered in any cultivars of tobacco Furthermore bothtobacco specific DNA based methods differ from those smellbased methods currently used in customs and could be usednot only for smuggling control but also for tariff classificationand for excise in tobacco trade In the future more tobaccoand nontobacco plants and more practical samples will betested to further confirm the specificity and applicability ofboth methods

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Sukumar Biswas and Wei Fan contributed equally to thiswork

Acknowledgments

This work was supported by the EU FP7 projectDECATHLON (613908) and the Programme of IntroducingTalents of Discipline to Universities (111 Project B14016)

References

[1] H Cao Y Wang Z Xie et al ldquoTGB the tobacco genetics andbreeding databaserdquo Molecular Breeding vol 31 no 3 pp 655ndash663 2013

[2] Euromonitor International [Database on the Internet]Cigarettes Global Euromonitor International 2015

[3] L Joossens and M Raw ldquoFrom cigarette smuggling to illicittobacco traderdquoTobacco Control vol 21 no 2 pp 230ndash234 2012

[4] Action on Smoking and Health ldquoIllicit trade in tobaccordquo ASHReport 2015 httpwwwashorgukfilesdocumentsASH 122pdf

[5] L Joossens D Merriman M Ross and M Raw HowEliminating the Global Illicit Cigarette Trade Would IncreaseTax Revenue and Save Lives The International Union AgainstTuberculosis and LungDisease Paris France 2009 httpglobaltobaccofreekidsorgfilespdfsenILL global cig trade full enpdf

[6] D D Layten and M T Nielsen Tobacco Production Chemistryand Technology Blackwell London UK 1999

[7] F Cholette and L-K Ng ldquoA real-time polymerase chain reac-tion (PCR) method for the identification of Nicotiana tabacumin tobacco productsrdquo Industrial Crops and Products vol 30 no3 pp 437ndash440 2009

[8] European Commission Directorate-General Taxation andCustomsUnion and Indirect Taxation and TaxAdministrationEnvironment and Other Indirect Taxes Excise Duty Tables PartIIImdashManufactured Tobacco REF 1035 REV 1 2012

[9] A-R Abbasi M Hajirezaei D Hofius U Sonnewald and LM Voll ldquoSpecific roles of 120572- and 120574-tocopherol in abiotic stressresponses of transgenic tobaccordquo Plant Physiology vol 143 no4 pp 1720ndash1738 2007

[10] W CMcCrone and J G Delly ldquoIndustrial dustsrdquo inTheParticleAtlas S Graft and A FeCoers Eds pp 213ndash550 Ann ArborScience Publishers Ann Arbor Mich USA 2nd edition 1973

[11] J Nagy ldquoTackling cigarette smuggling with enforcement casestudies reviewing the experience in Hungary Romania and theUnited Kingdomrdquo World Customs Journal vol 6 pp 29ndash402012

[12] Home Office UK Border Agency Tackling Tobacco SmugglingTogether 2008

[13] A Mahoney K La Londe T L Edwards C Cox B JWeetjens and A Poling ldquoDetection of cigarettes and othertobacco products by giant African pouched rats (Cricetomysgambianus)rdquo Journal of Veterinary Behavior Clinical Applica-tions and Research vol 9 no 5 pp 262ndash268 2014

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

[15] N Giermann M Schroder T Ritter and R Zrenner ldquoMolec-ular analysis of de novo pyrimidine synthesis in solanaceousspeciesrdquo Plant Molecular Biology vol 50 no 3 pp 393ndash4032002

[16] B Siegmund E Leitner andWPfannhauser ldquoDetermination ofthe nicotine content of various edible nightshades (Solanaceae)and their products and estimation of the associated dietarynicotine intakerdquo Journal of Agricultural and FoodChemistry vol47 no 8 pp 3113ndash3120 1999

[17] L Chen J Guo Q Wang G Kai and L Yang ldquoDevelopmentof the visual loop-mediated isothermal amplification assays forseven genetically modified maize events and their applicationin practical samples analysisrdquo Journal of Agricultural and FoodChemistry vol 59 no 11 pp 5914ndash5918 2011

[18] N Tomita Y Mori H Kanda and T Notomi ldquoLoop-mediatedisothermal amplification (LAMP) of gene sequences and simplevisual detection of productsrdquo Nature Protocols vol 3 no 5 pp877ndash882 2008

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Table 1 Descriptions of plant samples used in this study

Plant materials Description of the plant materials Providers

Nontobacco plants(fresh leaves)

Alfalfa (Medicago sativa) Altingia (Altingia gracilipes) canola (Brassica campestris)castor (Ricinus communis) Chinese jasmine (Jasminum officinale) coriander(Coriandrum sativum) Daphniphyllum (Daphniphyllum teijsmannii) eggplant

(Solanum melongena) garden petunia (Petunia times hybrid) Indica rice (Oryza sativavar indica) mondo grass (Ophiopogon japonicus) Pittosporum (Pittosporum

tobira) pomegranate (Punica granatum) sapodilla (Manilkara zapota) spinach(Spinacia oleracea) watermelon (Citrullus lanatus)

Locally collected

Dried tobacco leaves Burley (3) Oriental SAADI-6 Virginia (2) Oriental (5) reconstituted tobaccofoiled (1) Virginia grade A (1) Virginia grade B (1) Oriental BASMAK Bulgarian customs

Fresh tobacco leaves Burley Maryland Locally collected

Reconstituted tobaccoFactory sample I (tobacco stems no sulphate cellulose) factory sample II (notobacco stems no sulphate cellulose) factory sample III (tobacco stems with

sulphate cellulose) commercial wrapperDutch customs

Tobacco stem Tobacco stems Bulgarian customsCigarettes Liqun Double Happiness Mevius Marlboro Wuyeshen Locally purchasedThe numbers in brackets indicate the numbers of tobacco varieties of this species tested in the experiment

is therefore mainly based on the composition of the outerwrapper

Reconstituted tobacco is made from a pulp of mashedtobacco waste [6 7] The definition of reconstituted tobacco(Harmonised System World Customs Organisation) is asfollows ldquoit is made by agglomerating finely divided tobaccofrom tobacco leaves tobacco refuse or dust whether or noton a backing (eg sheet of cellulose from tobacco stems rdquoProducts with a wrapper of reconstituted tobacco whichmay consist partly of substances other than tobacco arealso seen as cigarscigarillos Because the differences ofexcise duties between cigarettes and cigars are significant inmany European countries [8] it is necessary to detect anyretail cigarscigarillos that do not match the definition of acigarcigarillo and should therefore be sold as a cigaretteTherefore a reliable method is needed to detect the presenceof tobacco in outer wrappers of these tobacco products

Tobacco identification is usually based on the detectionof the presence of nicotine neophytadiene [6] vitamin E[9] and microscopic fragments [10] Although these iden-tification methods are still used in certain cases (especiallyin reconstituted tobacco) they often failed to work [7] Inpractice microscopic tobacco fragments may not be detectedin reconstituted tobacco and on the other hand fraudulentactions may include the spraying or impregnation of nico-tine and neophytadiene into nontobacco constituents Thusalternative methods are required for successful detection andidentification of tobacco In EU customs officers use trainedanimals such as dogs [11ndash13] and giant African pouchedrats (Cricetomys gambianus) [13] to successfully detect thesmell of tobacco However because the odors of tobaccocan be readily enhanced or eliminated this method is likelyuseful to some extent for tobacco smuggling but obviouslynot applicable for tariff classification and for excise whichneedsDNAbasedmethods In a previous study a quantitativereal-time PCR (qPCR) assay based on a putrescine N-methyltransferase (PMT1) gene PMT1a was developed toidentifyNicotiana tabacum in tobacco products [7] However

because there is another PMT1b gene in tobacco genome andthe sequence of PMT1b was not available in the database [7]therefore the primers used for PMT1a are likely nonspecificto PMT1a In addition besides PMT1 3 additional PMT(PMT2ndashPMT4) genes with high similarity to each otherare present in tobacco genome [7] which makes the useof PMT1a as an endogenous reference gene of tobacco lessreliable

In this study by targeting a putative single copy tobaccoendogenous gene sequence of the uridine 51015840-monophosphatesynthase (UMPS) we developed two DNA based methodsa novel quantitative real-time PCR (qPCR) method and aloop-mediated isothermal amplification (LAMP) methodto detect the presence of tobacco components in variouscommodities The specificity and sensitivity were assayedusing various available materials including customs samplesreconstituted tobacco samples cured tobacco samples andlocally purchased cigarettes Our results showed that thenovel qPCR based on UMPS had 15-fold lower limit ofdetection and better specificity than the previously reportedPMT1a based qPCR method [7] Both qPCR and LAMPdeveloped on UMPS could be useful for rapid tobaccoidentification in a reliable and efficient way

2 Materials and Methods

21 PlantMaterials All the plantmaterials used in this studyincluding different tobacco and nontobacco materials andproducts are listed in Table 1

22 Genomic DNA Extraction and Purification A commer-cial DNA extraction kit the DNeasy Plant Mini kit (QiagenShanghai China) was used to extract genomic DNA 20mglyophilized tissue of different plant materials including freshtobacco leaves unprocessed tobacco reconstituted tobaccotobacco stems cigarettes and cigarette white wrapperswas considered for DNA isolation Briefly the sample was





4)2SO4 25mM













1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18M

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Sample ID

Ct

26252423

27

(c)




1 2 3 4 5 6 7 8 9

(a)

1 2 3 4 5 6 7 8 9M

(b)

DNA quantity (ng)

232425262728293031323334

Ct

Efficiency 92

12060301575375188094047

y = minus09118x + 33988

R2= 09981

012 024

(c)





1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19M

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Sample ID

2325272931333537

Ct

(c)




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

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14M

(b)

23252729313335

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

CtSample ID

(c)



4 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





4)2SO4 25mM













1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18M

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Sample ID

Ct

26252423

27

(c)




1 2 3 4 5 6 7 8 9

(a)

1 2 3 4 5 6 7 8 9M

(b)

DNA quantity (ng)

232425262728293031323334

Ct

Efficiency 92

12060301575375188094047

y = minus09118x + 33988

R2= 09981

012 024

(c)





1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19M

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Sample ID

2325272931333537

Ct

(c)




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

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14M

(b)

23252729313335

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

CtSample ID

(c)



4 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


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18M

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Sample ID

Ct

26252423

27

(c)




1 2 3 4 5 6 7 8 9

(a)

1 2 3 4 5 6 7 8 9M

(b)

DNA quantity (ng)

232425262728293031323334

Ct

Efficiency 92

12060301575375188094047

y = minus09118x + 33988

R2= 09981

012 024

(c)





1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19M

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Sample ID

2325272931333537

Ct

(c)




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

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14M

(b)

23252729313335

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

CtSample ID

(c)



4 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


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19M

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Sample ID

2325272931333537

Ct

(c)




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

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14M

(b)

23252729313335

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

CtSample ID

(c)



4 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

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