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-PCR evaluation for identification and sequence analysis ofot-and-mouth disease serotype O from 2006 to 2007 in Punjab,kistan

Saeed *, Qaiser Mahmood Khan, Usman Waheed, Memoona Arshad,hammad Asif, Muhammad Farooq

onal Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

ntroduction

Foot-and-mouth disease (FMD) is a highly contagiouseconomically important disease of cloven-hoofed

animals. The causative agent, foot-and-mouth diseasevirus (FMDV) belongs to the genus Aphthovirus of thefamily Picornaviridae [12].

RT-PCR procedures have been evaluated at the WRL forthe routine diagnosis of FMD virus using universal primersfor all seven serotypes [13] and serotype-specific primers[14], it is evaluated on an extensive range of field samplesand in most cases cell culture isolates or experimentallyinfected tissues rather than original field material had beenused. These studies showed that the sole use of RT-PCR was

T I C L E I N F O

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-and-mouth disease

type O

rse transcription-polymerase chain

tion

A B S T R A C T

FMD clinically positive 250 tissue samples (mouth and hoof epithelium and vesicle swabs,

tongue tissue) and 175 secretion samples (milk, saliva, serum, plasma) were evaluated by

RT-PCR for the diagnosis of FMD with different pair of universal and serotype-specific

primers from 2006 to 2007 in Punjab, Pakistan. Universal primer pair P1/P2 from VP1 gene

detected FMD in 182 out of 250 (72.8%) tissue and 92 out of 175 (52.6%) secretion samples,

while universal primer 1F/1R from 50UTR region detected FMD in 218 out of 250 (87.2%)

tissue and 142 out of 175 (81.1%) secretion samples. 1F/1R proved better than the P1/P2

primer pair for primary diagnosis of FMD, direct from the clinical positive samples. Direct

sequencing of the Universal primer pair P1/P2 revealed that O serotype of FMD was

circulating in this region. O serotype of FMD was detected with O-1C(ARS4)/PNK 61,

AU(O)/AU(rev), AU(O)/PNK61 primer pairs, these primer pairs also compared with each

other. AU(O)/AU(rev) and AU(O)/PNK61 detected O serotype of FMD in 88.9% tissue and

swab (mouth and hoof vesicle swabs) samples and 71.9% different secretion (Milk, Saliva,

Serum, Plasma) samples, while O-1C(ARS4)/PNK 61 detected 48.1% tissue and swab

(mouth and hoof vesicle swabs) samples and 37.5% different secretion (Milk, Saliva, Serum,

Plasma) samples. AU(O)/AU(rev), AU(O)/PNK61 primer pairs detected 40.8% more tissue

and swab samples, while these pairs detected 34.4% more secretion samples. Cloning of

PCR product of AU(O)/AU(rev) VP1 gene and sequencing for phylogenetic studies revealed

that O serotype of FMD circulating in Punjab, Pakistan was very diverse genetically from

the ‘O’ serotype in Middle East and Europe respectively. The dendrogram showed that

Pakistan ‘O’ serotype was very much similar genetically to its neighbor countries (Sri

Lanka, India, Iran, Iraq, and China) and Pan Asia 1 linage which cause 2001-outbreak in UK

and 1994-outbreak in Saudi Arabia, etc.

� 2010 Elsevier Ltd. All rights reserved.

Corresponding author.

E-mail addresses: alisaeednibge@gamil.com (A. Saeed),

@yahoo.com (Q.M. Khan), dr.usman.waheed@gmail.com

aheed), moonaarshad@gmail.com (M. Arshad),

nibge@hotmail.com (M. Asif), farooq.dvm@gmail.com (M. Farooq).

Contents lists available at ScienceDirect

Comparative Immunology, Microbiologyand Infectious Diseases

journa l homepage: www.e lsev ier .com/ locate /c imid

ease cite this article in press as: Saeed A, et al. RT-PCR evaluation for identification and sequence analysis of foot-and-outh disease serotype O from 2006 to 2007 in Punjab, Pakistan. Comp Immunol Microbiol Infect Dis (2010),

oi:10.1016/j.cimid.2009.10.004

7-9571/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.

10.1016/j.cimid.2009.10.004

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inappropriate for primary diagnosis of FMD virus. There-fore, RT-PCR procedures had not demonstrated sufficientlyfor diagnosis of FMD virus [15].

Since, success of any PCR is dependent on the efficiencyof primer and template binding, it would be ideal if theprimers were designed on the virus sequences native tothat particular geographical area [8]. Within the FMDVgenome, the P1 region that codes for structural (capsid)proteins is more variable than those codes for non-structural proteins. Further, it has been reported thatsero- and subtype specificity lies within the P1 region,more particularly in the 1D gene [7].

The molecular techniques such as reverse transcrip-tion-polymerase chain reaction, nucleotide cyclic sequenc-ing and phylogenetic analyses have become valuable toolsfor the characterization and epidemiological tracing ofFMD outbreaks. Phylogenetic analyses have been mostlybased on sequences from the VP1-coding region [5].

Spatial distribution of foot-and-mouth disease inPakistan was predicted using outbreaks reported to OfficeInternational des Epizootics (OIE), density of livestock, anda probability co-kriging analytic method. The areas athighest risk were located throughout the Punjab regionconnecting India and Afghanistan, which may be aconsequence of increased contact with infectious animalsmoving in to the region from the other FMD-endemiccountries. The empirical distribution of outbreaks includedas covariate; whereas inclusion of the small ruminantsdistribution did not significantly improve the predictionability of model. It may be due to a higher likelihood thatinfected cattle will show clinical sign, compared with smallruminants, which would result in a differentional under-reporting of small ruminant cases of FMD [11].

This study was designed for the development andevaluation of RT-PCR on original field material. It alsoincludes the comparison of universal primer pairs andserotype-specific primer pairs for the diagnosis of FMDwith designed and already reported serotype-specificprimer pairs. It is envisaged that this present evaluatione.g. alternative use of primer, sequence analysis will help inthe future design of improved PCR protocols, for exampleprimer selection, multiplex PCR development, nested PCRformats and molecular epidemiology of FMD. This study

will also enhance the sensitivity of virus detection in theprimary diagnosis of FMD direct from the field sample. Thesequence analysis will help to understand the molecularnature of virus circulating in this region. It will help for theselection of vaccination and strategies for the control ofFMD in this region.

2. Materials and methods

FMD clinically positive 425 samples were collectedduring 2006–2007 from 10 outbreaks from different areas(Faisalabad, Sargodha, Jhang) of Punjab, Pakistan whichinclude 250 tissue/swab samples (220 mouth vesicles, 24hoof vesicles, 6 tongue tissue) and 175 different secretionssamples (25 milk, 50 saliva, 50 serum, 50 plasma) as shownin Table 1 from the different type of animals e.g. cattle,buffalo and goat. The swab samples were collected withear cleaning cotton sterile buds directly from the site ofvesicle (mouth, hoof, tongue) into sterile falcon tube with1.5 ml of transport medium (equal amounts of glycerol and0.04 M phosphate buffer having the pH 7.2, and thePenicillin at the final concentration of 1000 IU/ml), tissuesamples directly into transport medium and blood indifferent BD Vacutainer1 (blood containing tube with andwithout heparin), finally transported to animal virologylaboratory NIBGE, on ice.

The RNA was extracted with TRIzol Method (standardmethod as per manufacturer) (GibcoBRL, Sao Pablo, Brazil).Reverse transcription was performed with Random Hex-anucleotide primers as per supplier’s (MBI Fermentas,Graiciunau 8, Vilmius 2028, Lithuania) and M-MuLVRTase(MBI Fermentas) by using standard reaction condi-tions (a 10 ml volume containing 5 ml RNA solution (1–5 mg of RNA), 2 ml of Random hexanucleotide primer(50 ng/ml), 3 ml DEPC). Then 10 ml of the RT-mix was addedin it which contained, 4 ml of 5� RT buffer (Tris–HCl50 mM, pH 8.3), 2 ml of DTT (10 mM), 2 ml of BSA (0.1 mg/ml), 1 ml dNTPs (10 mM) (each) and 2 ml of M-MuLV RT(200 U). It was incubated at room temperature for 5 min,then at 37 8C/60 min. The detail of primer sequences usedin this study is given in Table 2.

The thermocycling was performed in thermal cycler(Master Cycler Gradient, Eppendorf) with standard reac-

Table 1

Primer pairs, sample collection during 2006–2007 (total sample processed, tissue/swab sample processed, secretion samples processed).

Primer pairs Total samples

processed

Sample collection during 2006–2007

Tissue and swab samples processed Secretion samples processed

Total M.V.S. H.V.S. T.T. Total Milk Saliva Serum Plasma

Total samples processed

with universal primers

425** 250a 220 24 6 175a 25 50 50 50

P1/P2 detected 274 182 (72.8%) 172 6 4 92 (52.6%) 5 (20%) 35 23 29

1F/1R detected 360 218 (87.2%) 198 14 6 142 (81.1%) 16 (64%) 46 40 40

Total samples processed

with serotype-specific

primers

59** 27a 22 3 2 32a 6 12 7 7

O-1C(ARS4)/PNK61 detected 25 13 (48.1%) 12 1 0 12 (37.5%) 2 (33.3%) 5 2 3

AU(O)/AU(rev) detected 47 24 (88.9%) 20 3 1 23 (71.9%) 4 (66.6%) 9 4 6

AU(O)/PNK61 detected 47 24 (88.9%) 20 3 1 23 (71.9%) 4 (66.6%) 9 4 6

Bolds are the main values and non-bold were the subdivisions of the main values in both sides (e.g. in rows & in columns).a M.V.S., mouth vesicle swab; H.V.S., hoof vesicle swab; T.T., tongue tissue.** Total number of samples processed for diagnosis.

Please cite this article in press as: Saeed A, et al. RT-PCR evaluation for identification and sequence analysis of foot-and-mouth disease serotype O from 2006 to 2007 in Punjab, Pakistan. Comp Immunol Microbiol Infect Dis (2010),doi:10.1016/j.cimid.2009.10.004

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Pr

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A. Saeed et al. / Comparative Immunology, Microbiology and Infectious Diseases xxx (2009) xxx–xxx 3

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conditions (a 50 ml volume containing 5 ml of 10� PCRfer (containing Tris–HCl 200 mM, KCl 500 mM, MgCl2

mM), 2.5 ml of MgCl2 (25 mM), 1 ml of dNTPs (10 mM)ch), 1 ml Primer (each sense/anti-sense) (10 pm/ml),ml of Taq Polymerase, 35 ml of DEPC treated water andl of cDNA). The thermocycling profile was 94 8C formin/1 cycle, 94 8C for 01 min/30 cycles, 54 8C formin/30 cycles (for universal) and 58 8C for 01 min/30les (for ‘O’ serotype-specific) primer pairs, 72 8C formin/30 cycles and final extension at 72 8C for 10 min/01le.1.5% of agarose gel was prepared in electrophoresisaratus (GibcoBRL, Life Technologies) and 100 bps ladderI Fermentas, Graiciunau 8, Vilmius 2028, Lithuania) was

d to identify the band. Afterwards, the gel was checkeder UV Trans-illuminator (MacroVue, UV20, Hoefer,er Lourmat, France) to record the results.

PCR product was ligated in pTZ57R vector (MBImentas). The reaction mixture with final volume ofml (1 ml of pTZ57R, 7 ml of PCR product, 2 ml of ligationfer, 1 ml of T4 DNA Ligase, 1 ml of PEG4000 and 9 ml of

2O) incubated overnight at 16 8C. Ligated productsformed into Escherichia coli in Top10 alpha by heat

ck method. The screenings of recombinant E. coli clonesre done by using the Plamid Alkaline Extraction Method

20 ml of the reaction mixture (5 ml of plasmid DNA,l of 10� reaction buffer Y+, 0.5 ml each of the restrictionyme e.g. Pst1 and EcoR1, 1 ml of RNase and 11 ml ofble distilled H2O) incubated at 37 8C for 1 h. Theples having the correct inserted fragment werected and marked as recombinant clones on 1% agarose.

er screening the selected clone, plasmid was isolated bye JET Plasmid Miniprep Kit (MBI Fermentas). Then,ated plasmid was sequenced from Macrogen (Korea).Phylogenetic tree was constructed by using clus-1.81.msw, Tree View Software (Intall Sheild Corpora-, Inc., version 3.0.111.0) and PAUP 4.0b4 computer

grams. 200 nt was selected because AU(O)/AU(rev)plify only 200 nt from VP1 gene which was 31.2% of thel VP1 gene sequence.

3. Result

Total 250 tissue/swabs and 175 secretion samplesrepresentative of different outbreaks were tested withuniversal primer pairs P1/P2 and 1F/1R (Table 1).

P1/P2 primer pair detected FMDV directly into 182 outof 250 tissue and swab samples, whereas 92 out 175different secretion samples. It detected 172 out of 220mouth vesicle swab samples, 6 out of 24 hoof vesicle swabsamples and 4 out of 6 tongue tissue samples whereas 5out of 25 milk samples, 35 out of 50 saliva samples, 23 outof 50 serum samples, 29 out of 50 plasma samples(Table 1).

1F/1R primer pair detected FMD direct into 218 out of250 tissue and swab samples, whereas 142 out 175different secretion samples. It detected 198 out of 220mouth vesicle swab samples, 14 out of 24 hoof vesicleswab samples and 6 out of 6 tongue tissue sampleswhereas 16 out of 25 milk samples, 46 out of 50 salivasamples, 40 out of 50 serum samples, 40 out of 50 plasmasamples (Table 1).

Direct sequencing of P1/P2 PCR products and NCBI Blastanalysis showed O serotype of FMDV. 27 representativetissue/different swab samples and 32 different secretionsamples were selected for the confirmation of O serotypeof FMD with serotype-specific primer pairs.

RT-PCR with serotype-specific primer pairs (O-1C(ARS4)/PNK61), AU(O)/AU(rev) and AU(O)/PNK61(Table 2) confirmed the presence of O serotype of FMDV.O-1C(ARS4)/PNK61 detected O serotype of FMDV in 13 outof 27 tissue/different swab samples which included 12 outof 22 mouth vesicle swab samples, 1 out of 3 hoof vesicleswab samples, 0 out of 2 tongue tissue samples. It detected12 out of 32 secretion samples which include 2 out of 6milk samples, 5 out of 12 saliva samples, 2 out of 7 serumsamples, 3 out of 7 plasma samples (Table 1).

AU(O)/AU(rev) primer pair detected O serotype ofFMDV in 24 out of 27 tissue/different swab samples whichinclude 20 out of 22 mouth vesicle swab samples, 3 out of 3hoof vesicle swab samples, 1 out of 2 tongue tissue

le 2

ences used in reactions, sense of primers, gene of attachment, product size, and type of primer.

imers Sequence (50–30) Sense Gene of

attachment

Product size

(bp)

Type of primer

50-CCTACCTCCTTCAACTACGG-30 + 1D 216 Universal primers (all seven

serotypes of FMD)50-CTCAGGTTGGGACCCGGGAAG-30 � PB2A

50-GCCTGGTCTTTCCAGGTCT-30 + 50UTR 328 Universal primers (all seven

serotypes of FMD)50-CCAGTCCCCTTCTCAGATC-30 � 50UTR

1C(ARS4) 50-ACCAACCTCCTTGATGTGGCT-30 + 1C 1301 ‘O’ serotype specific

K61 50-GACATGTCCTCCTGCATCTG-30 � 2B

U(O) 50-CAAGTGTTGGCCCAGAAGG-30 + 1D 623 ‘O’ serotype specific

U(rev) 50-GGGGCTCCGAAACTGAAGA-30 � 2B

U(O) 50-CAAGTGTTGGCCCAGAAGG-30 + 1D 320 ‘O’ serotype specific (nested set

of primer ‘O’ serotype specific)K61 50-GACATGTCCTCCTGCATCTG-30 � 2B

13-f (20) 50-GTAAAACGACGGCCAGT + Primer use in sequencing

13-R (20) 50-GCGGATAACAATTTCACACAGG �

2, 1F/1R and O-1C(ARS4)/PNK61, Reid et al. [15]; *AU(O), *AU(rev) was self-designed.

ease cite this article in press as: Saeed A, et al. RT-PCR evaluation for identification and sequence analysis of foot-and-outh disease serotype O from 2006 to 2007 in Punjab, Pakistan. Comp Immunol Microbiol Infect Dis (2010),

oi:10.1016/j.cimid.2009.10.004

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samples. It detected 23 out of 32 secretion samples whichincluded 4 out of 6 milk samples, 9 out of 12 saliva samples,4 out of 7 serum samples, 6 out of 7 plasma samples(Table 1).

AU(O)/PNK61 nested primer pair detected O serotype ofFMDV in 24 out of 27 tissue/different swab samples whichinclude 20 out of 22 mouth vesicle swab samples, 3 out of 3hoof vesicle swab samples, 1 out of 2 tongue tissuesamples. It detected 23 out of 32 secretion samples whichincluded 4 out of 6 milk samples, 9 out of 12 saliva samples,4 out of 7 serum samples, 6 out of 7 plasma samples(Table 1).

Phylogenetic analysis revealed that FMDV circulating inthis region was variable from European and Middle Eastisolates respectively. But it was similar to its neighbor and2001 UK isolates (Figs. 1 and 2).

4. Discussion

Foot-and-mouth disease remains endemic in manyregions of the world. But its control in Asian countries isvery difficult because these countries have a large numberof sheep, cattle population, poverty and uncontrolledmovement of these animals. It has always been the mostimportant constraint to international trade in live animalsand animal products as World Trade Organization agree-ments remove other obstructions. The imposition of banson the movement of susceptible livestock following thediscovery of an outbreak is deemed necessary to preventthe spread of highly contagious disease [9,10,18]. There-

fore timely diagnosis is very important to ban themovement of infected animals.

Universal and serotype-specific primer sets were usedin simple reverse transcription-polymerase chain reaction(RT-PCR) assays on field samples of epithelium andvesicular fluid to determine their suitability for primarydiagnosis of all seven serotypes of foot-and-mouth disease(FMD). Universal primer sets, located principally in the P1capsid-coding region, were generally inferior to the 5% UTRuniversal primer set (1F/1R). 1F/1R primer can be used forthe primary diagnosis of FMD along with other diagnosticmethods but it is not efficient for all strains of FMDV (SAT1/2/3). It is envisaged that this evaluation and alternativeuse of primers will lead to the development of novel PCRstrategies, for example nested PCR formats, with concomi-tant improvement in the speed of primary diagnosis ofFMD [15]. In this study, evaluation and alternative use ofalready available and newly designed universal andserotype-specific primer sets in RT-PCR e.g. nested format,improved the primary and serotype-specific diagnosis ofFMDV on variety of field samples.

The results in this study are consistent with Reid et al.[15] that 1F/1R proved better for the primary diagnosis ofFMDV. But it evaluation direct on field samples wereunique feature of this study. 1F/1R detected FMDV in 87.2%tissue and epithelium swab samples (Table 1), while P1/P2detected 72.8% of the tissue and epithelium swab samples.1F/1R detected 14.4% more tissue and epithelium swabfield samples. 1F/1R detected FMDV in 81.1% secretionsamples (Table 1), while P1/P2 detected 52.6% of the

Fig. 1. Phylogenetic tree of ‘O’ serotype of FMDV, 31.2% of VP1 sequence with bootstrap replication (100 bootstraps) and rooted by Pak Sar3 goat

(AM942749).

Please cite this article in press as: Saeed A, et al. RT-PCR evaluation for identification and sequence analysis of foot-and-mouth disease serotype O from 2006 to 2007 in Punjab, Pakistan. Comp Immunol Microbiol Infect Dis (2010),doi:10.1016/j.cimid.2009.10.004

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retion samples. 1F/1R detected 28.5% more secretiond samples. Primary diagnosis with P1/P2 was alsoificant because 1F/1R set PCR products lacks geneticrmation. Direct sequencing of PCR product of P1/P2 setniversal primers and BLAST analysis at NCBI showed

t ‘O’ serotype was circulating.Serotype O was isolated from Pakistan, Afghanistan, Srika, Vietnam while serotypes O, A and Asia 1 remainsemic in India, parts of China, Bangladesh and Thailand]. O-1C(ARS4)/NK61 primer pair was specific to Ootype but had low sensitivity. It detected viral RNA in

than half of epithelium suspensions containing type Os. Nested system improved the RT-PCR performance on

thelial suspensions of each serotype [15]. Similarly instudy, O-1C(ARS4)/NK61 detected O serotype in less

n half of tissue/epithelium swab samples and secretionples (48.1% tissue and epithelium swab samples and

5% secretion samples). O-1C(ARS4)/NK61 targeted ag fragment from the FMDV genome and geneticiation of Asian isolates [5] may be the reasons that

of primer remained unsuccessful to diagnose ‘O’otype in many samples. AU(O)/AU(rev) designed froml isolates and AU(O)/PNK61(nested) primer pairsroved detection limit of O serotype into same samplesthese both pairs equally detected O serotype in 88.9%ue and epithelium swab samples and 71.9% secretionples (Table 1). AU(O)/AU(rev) and AU(O)/PNK61er pair detected 40.8% more tissue/ epithelium

b samples and 34.4% more secretion samples thanC(ARS4)/NK61.

Milk is an ideal medium for laboratory diagnosis of FMDmay be particularly appropriate for the surveillance of

disease in dairy herds. Milk has been used to monitorinfections and to identify herds with infected individualsfor other viral diseases [2,3]. Previously real-time RT-PCR(rRT-PCR) is used as diagnostic tool for the laboratorydetection of FMDV in vesicular epithelial tissue [16,19],and in serum, nasal swabs and oesophageal–pharyngealscraping (‘‘probang’’) samples [20]. rRT-PCR detectedFMDV in milk up to 23 days post-inoculation which waslonger than Virus Isolation [17]. But in this study, first timemilk samples were directly tested with RT-PCR which wasa useful alternative to the real-time PCR technique due toits dual importance in diagnosis of FMDV and sequenceanalysis of the amplicons. All these samples were collectedwhen the animals were suffering and showing clinical signof FMD at different incubation periods. 25 representativemilks samples tested with RT-PCR which detected 20%with P1/P2, 64% with 1F/1R, 33.3% with O-1C(ARS4)/PNK61, 66.6% with AU(O)/AU(rev) and 66.6% with AU(O)/PNK61 primer pairs (Table 1). The alternative use of primere.g. nested PCR formats and different combination ofprimer pairs were improved primary as well as serotype-specific diagnosis of FMDV.

The nucleotide sequence encoding the VP1 capsidprotein of FMDV alone have provided valuable insight intothe emergence of various strains and serotypes worldwide[1,5]. Pan Asian topotype was imported into Saudi Arabiaby infected livestock then spread throughout Middle East.It is still present in this region and has replaced all otherstrains of serotype ‘O’ [4]. The Blast analysis andphylogenetic analysis (Figs. 1 and 2) of 200 nt from VP1(31.2%) gene of FMD serotype ‘O’ circulate during 2006–2007-outbreaks in Pakistan showed that it was similar to

Fig. 2. Phylogenetic neighbor joining tree of ‘O’ serotype of FMDV.

ease cite this article in press as: Saeed A, et al. RT-PCR evaluation for identification and sequence analysis of foot-and-outh disease serotype O from 2006 to 2007 in Punjab, Pakistan. Comp Immunol Microbiol Infect Dis (2010),

oi:10.1016/j.cimid.2009.10.004

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Pan Asian topotype of FMDV ‘O’ serotype which has caused2001-outbreaks in UK, 1994-outbreak in Saudi Arabia andprevious outbreaks in India. The serotype isolated fromPakistan was highly variable from the ‘O’ serotype isolatesof other European countries, less variable from Middle Eastand similar to PanAsia1 lineage of ‘O’ serotype of FMD of2001-outbreak of UK and other neighbor countries.

The combination of 1F/1R and P1/P2 are best set ofprimer for primary and initial serotype diagnosis of FMDdirect in clinical samples. AU(O)/AU(rev) are the better setof primer for the diagnosis of ‘O’ serotype of FMD alongwith AU(O)/PNK61 in tissue/epithelium swab and secre-tion samples. O-1C(ARS4)/PNK61 is an important set ofprimer for molecular epidemiological work because ittargeted a long portion of genome. Pan Asia 1, O serotype ofFMD circulated in this region which replaced most of theexisting strain of FMD. More molecular work is required forthe diagnosis of FMD with variety of primer pairs withdifferent combinations on variety of samples and to studythe molecular nature of circulating virus. In regard ofepidemiology, continuous work is required which will helpto make strategies for the control of FMD in this region e.g.development of multiplex PCR, selection of vaccine and tomonitor the phylogenetic relations with other isolates.

Acknowledgement

Author wants to acknowledge the scientists of PlantBiotechnology, NIBGE, Faisalabad for their help in Phylo-genetic work.

References

[1] Bronsvoort BM, Radford AD, Tanya VN, Nfon C, Kitching RP, MorganKL. Molecular epidemiology of foot-and-mouth disease viruses inthe Adamawa province of Cameroon. J Clin Microbiol 2004;42:2186–96.

[2] Drew TW, Yapp F, Paton DJ. The detection of bovine viral diarrhoeavirus in bulk milk samples by the use of a single-tube RTPCR. VetMicrobiol 1999;64:145–54.

[3] Heath GS, King DP, Turner JLE, Wakeley PR, Banks M. Use of aninternal standard in a TaqMan1 nested reverse transcriptionpoly-merase chain reaction for the detection of bovine viral diarrhoeavirus. Vet Microbiol 2003;96:357–66.

[4] Knowles NJ, Samuel AR, Davies PR, Kitching RP. Donaldson AIoutbreak of foot-and-mouth disease virus serotype O in the UKcaused by a pandemic strain. Vet Rec 2001;148:258–9.

[5] Knowles NJ, Samuel AR. Molecular epidemiology of foot and mouthdisease virus. Virus Res 2003;91:65–80.

[6] Birnboim HC, Doly J. A rapid alkaline extraction procedure forscreening recombinant plasmid DNA. Nucleic Acids Res 1979;24(7(6)):1513–23.

[7] Cheung A, De LaMarter J, Weiss S, Kupper H. Comparison of the majorantigenic determinants of different serotypes of foot-and-mouthdisease virus. J Virol 1983;48:451–9.

[8] Giridharan P, Hemadri D, Tosh C, Sanyal A, Bandyopadhyay SK.Development and evaluation of a multiplex PCR for differentiationof foot-and-mouth disease virus strains native to India. J VirolMethods 2005;126:1–11.

[9] Kitching RP. A recent history of foot-and-mouth disease. J CompPathol 1998;118:89–108.

[10] Kitching RP. Foot-and-mouth disease: current world situation. Vac-cine 1999;17:1772–4.

[11] Perez AM, Thurmond MC, Carpenter TE. Probability co-kringingestimation of foot and mouth disease spatial distribution in Paki-stan. In: GisVet Draft conferences presentations; 2004, www.gisvet.org/Conferences_2004_Presentations.

[12] Racaniello VR. Picornaviridae: the viruses and their replication. In:Fields virology3rd ed., Lippincott Williams and Wilkins; 2001 . p.658–722.

[13] Reid SM, Forsyth MA, Hutchings GH, Ferris NP. Comparison ofreverse transcription polymerase chain reaction, enzyme linkedimmunosorbent assay and virus isolation for the routine diagnosisof foot-and-mouth disease. J Virol Methods 1998;70:213–7.

[14] Reid SM, Hutchings GH, Ferris NP, De Clercq K. Diagnosis of foot-and-mouth disease by RT-PCR: evaluation of primers for serotypiccharacterisation of viral RNA in clinical samples. J Virol Methods1999;83:113–23.

[15] Reid SM, Ferris NP, Hutchings GH, Samuel AR, Knowles NJ. Primarydiagnosis of foot-and-mouth disease by reverse transcription poly-merase chain reaction. J Virol Methods 2000;89:167–76 [Shortcommunication].

[16] Reid SM, Grierson SS, Ferris NP, Hutchings GH, Alexandersen S.Evaluation of automated RT-PCR to accelerate the laboratory diag-nosis of foot-and-mouth disease virus. J Virol Methods 2003;107:129–39.

[17] Reid SM, Parida S, King DP, Hutchings GH, Shaw AE, Ferris NP, et al.Utility of automated real-time RT-PCR for the detection of foot-and-mouth disease virus excreted in milk. Vet Res 2006;37:121–32.

[18] Schley D, Gubbins S, Paton DJ. Quantifying the risk of localisedanimal movement bans for foot-and-mouth disease. PLoS ONE2009;4(5):e5481. doi: 10.1371/journal.pone.0005481.

[19] Shaw AE, Reid SM, King DP, Hutchings GH, Ferris NP. Enhancedlaboratory diagnosis of foot-and-mouth disease by real-time poly-merase chain reaction. Rev Sci Tech Off Int Epizoot 2004;23:1003–9.

[20] Zhang Z, Alexandersen S. Detection of carrier cattle and sheeppersistently infected with foot-and-mouth disease virus by a rapidrealtime RT-PCR assay. J Virol Methods 2003;111:95–100.

Please cite this article in press as: Saeed A, et al. RT-PCR evaluation for identification and sequence analysis of foot-and-mouth disease serotype O from 2006 to 2007 in Punjab, Pakistan. Comp Immunol Microbiol Infect Dis (2010),doi:10.1016/j.cimid.2009.10.004