Development and Optimization of Multiplex PCR for the Identification of A , O and Asia – 1 Serotypes of FMDV in Pakistan

Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan; Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan; Department of Epidemiology and Public Health; University of Veterinary and Animal Sciences, Lahore, Pakistan; Livestock and Dairy Development Department, Government of Punjab, Pakistan; Chittagong Veterinary and Animal Sciences University Chittagong, Khulshi–4225, Bangladesh *Corresponding author: ahadvet1969@yahoo.co.uk


INTRODUCTION
Foot and mouth disease is a highly contagious viral disease of cloven hoofed animals.The virus belongs to picornaviridae family.It is single stranded, positive sense naked virus.The symmetry of the virus is icosahedral.Seven serotypes have been identified which are designated as Asia1, O, A, C, SAT 1 SAT 2 and SAT3.Since 2004, type C has not been reported from anywhere in the world.O and A serotypes found almost word wide and type Asia-1 circulated in the south Asia.SAT1, SAT2, SAT3 have been reported in African territory (Hall et al., 2013;Sangula et al., 2011;Knowles and Samuel, 2003).The disease is endemic in Pakistan throughout the year with major serotypes A, O and Asia1.It causes a loss of 6 billion rupees approximately to farmers annually in Pakistan (Anjum et al., 2006).
FMD virus can be isolated by using BHK 21 and calf kidney cells.It can be diagnosed by viral neutralization test (OIE, 2012).Monoclonal antibody based direct ELISA (MSD-ELISA) can detect FMDV antigen.This technique can be used to identify serotype.Complement fixation test is also used for the serotyping of FMDV.Besides these techniques, in the past few years reverse transcriptase polymerase chain reaction (RT-PCR) has been used for the diagnosis of FMD (Xu et al., 2013).The mRT-PCR for the diagnosis of FMDV is gaining popularity because of its high sensitivity and specifity (Hindson et al., 2008).The RT-PCR is specific, rapid and sensitive method for typing and detection of the FMDV as well as for its differentiation from other vesicular diseases like Bovine Viral Diarrhea and Vesicular Stomatitis Virus (Callens and Clercq 1997).RT-PCR successfully detects small amounts of the FMD virus in biological samples (Wiesław and Kęsy, 2010).In this study we optimize mRT-PCR in early detection of foot and mouth disease virus.

Virus Stock
Viruses A, O and Asia1 serotypes of FMDV were obtained from culture bank of Quality Operation Laboratory at University of Veterinary and Animal Sciences, Lahore.according to manufacturer instructions.Extracted RNA was immediately used for cDNA synthesis (Chomczynski and Sacchi, 1987).

cDNA Synthesis
To prepare cDNA from extracted RNA, the RevertAid M-MuLV First Strand cDNA Synthesis Kit (# K1622, Fermantas) was used following the recommendations of Giridharan et al. (2005).

Primer Design
Genomic

Optimization of Multiplex PCR
Separate PCR reaction for A, O and Asia1 was carried out in a reaction mixture of 25 μl volume using kit (K#0211, Fermentas) with serotype specific primers.Universal primer pair IF: IR was used for genome detection of FMDV (Reid et al., 2000).Multiplex PCR was performed on combination of three serotypes (A, O and Asia1) at different annealing temperatures ranging from 55 0 C to 57°C.Reaction mixture containing 9 μl cDNA, 1.5μl (0.3μM) of each forward and reverse primer total 9 μl for three serotypes (for VP1 gene A, O and Asia1), 20μl of PCR master mix (Taq DNA-Polymerase 0.05 units/ μl, MgCl 2 4 mM and dNTPs 0.4 mM) and MB grade-H 2 O 12 μl to the final volume 50 μl.
The concentration was changed by using 1μl of, 2μl and 3μl of template DNA and 0.5μl, 1μl and 1.5μl of primers for each serotype.Thermocycler was programmed as; denaturation at 94°C for 7 minutes one cycle, 94°C for 1 minute, annealing at 56 0 C for 1 minute and extension at 72 °C for 1 minute 35 cycles and final extension at 72°C for 10 minutes one cycle (Callens and Clercq1997).
Multiplex PCR products were analyzed on a 3% agarose gel in 1 μg/ ml Ethidium Bromide staining solution (Tosh et al., 2002).To differentiate between the bands of O and Asia1 multiplex PCR products were resolved on 7% polyacrylamide gel electrophoresis (PAGE) at 120 volts for 8 hours.

Sensitivity and Specificity
To study the sensitivity of multiplex PCR (mPCR) a series of 10-fold serial dilution of extracted RNA of each serotype was carried out.The sensitivitty of PCR for each serotype was optimized by making 10-fold serial dilutions of extracted RNA.Sensitivity of mPCR was also optimized at different primer to template concentrations.The primer to template ratio was optimized by using 0.5 μl, 1 μl, 1.5 μl and 1.5 μl, 2 μl, 3 μl respectively for each primer.Annealing temperature of primers for all the three serotypes of FMDV was ranging from 53 0 C -60°C.Primers were also run with peste des petits ruminants (PPR) virus RNA which was extracted from PPR vaccine.

Application of Multiplex PCR on Field Samples
The samples were collected from 20 infected cattle and buffaloes from Kasur and Lahore districts of Punjab, Pakistan.The samples were the tongue epithelial tissue and ruptured vesicles of clinically infected animals.The samples were collected in the transport media 10% PBS-glycerol solution having pH of 7.2.The samples were stored at -40 °C until analysis.The optimized mPCR was used to detect the prevalent serotypes in the field.

RESULTS
Universal primer pair (IF: IR) was used and it was shown that it can detect FMDV genotype by yielding an amplicon of 328 bp (Figure 2.0).Separate reactions for A, O and Asia1 serotypes were carried out with serotype specific primers.mRT-PCR was developed to detect A, O and Asia1 serotypes of FMDV in a single reaction through 35 cycles of PCR.The mRT-PCR products were 386 bp for A, 240 bp for Asia1and 232 bp for O (Figure 1).
Out of 20 field samples 18 were found to be positive with Asia1 serotype.This showed 100% sensitivity of PCR.Sensitivity of mPCR was also optimized at a template concentration of 3 μl and 1.5 μl (0.3 μM) of each forwad and reverse primer.Dilutions of RNA from 10 1 -10 3 were successfully amplified by PCR.While dilution 10 4 showed bands with very weak intensity and dilution 10 5 has given no results.
Specificity of FMDV primers was determined on PPRV (Figure 3 Lane 3).Non specific band were observed in case of PPRV RNA.mRT-PCR was found to be a specific assay for type A, O and Asia1 serotypes of FMDV.This technique was also analysed on field samples detecting serotype Asia1 (Figure 3).Amplicons were analyzed on 3% PAGE to visualize more clear bands of serotype O and Asia1 which is shown in figure 4.

DISCUSSION
In this study we found mPCR a very quick and reliable method to identify the specific serotype of FMD in any filed outbreak.A 328 bp fragment was amplified for all three serotypes (A, O and Asia1) using universal primer pair (IF: IR).Similarly a study was carried out by Reid et al (2000) using same primers (1F:1R); their results had also an amplicon of 328 bp.Alamdari et al. (2006) used phenolchloroform thiocyanate-based method for RNA extraction rather than trizole reagent.Giridharan et al., (2005) isolated RNA using kit (Qiagen, Germany).The difference in RNA extraction protocol has been found to be no effect on the results.For cDNA synthesis instead of random hexamers they used oligonucleotide primers.Their results were different giving a band size of 131 bp.Difference in results was due to the use of different primer pair for genome detection of FMDV.For serotype identification and primers optimization of A, O and Asia1, PCR was carried out using serotype specific primers designed against VP1 gene in individual reaction mixture of 25 μl (A1F:A1R, O1F:O1R, AS1F:AS1R).Amplicon size of 386, 232 and 240 bp for A, O and Asia1 serotypes were obtained respectively in current study.Giridharan et al. (2005) designed primers VP1 gene native to their country (serotype specific) and capsid-coding region of FMDV.Their results for O, A, Asia1 were 249, 376 and 537 bp respectively.Difference was due to the primers' positions giving different amplicons as compared to the present study.
Then optimization of PCR cycling conditions for A, O and Asia1 serotypes of FMDV done individually.Among the cyclic conditions, increase of annealing temperature from 56 to 60°C did not alter the amplification efficiency but above 60°C, it was affected adversely giving non specific products.At primer to template concentration/ratio of 0.5μl, 1μl and 1.5μl, to 1.5μl, 2μl and 3μl template, product was amplified at primer concentration of 1.5μl (forward and reverse) and template concentration of 3.0μl whereas; in other combinations non specific products were obtained.The number of cycles were only increased from 30-40 for that template DNA which has higher GC contents.
Callens and De Clercq (1997) used primers for the amplification of A, O and Asia1 serotypes of FMDV.Primers' sequences were different from the present study.They designed from the 1D and 2AB genes of 1C region of the FMD viral genome.Primers amplified the fragments 402, 732and 296 bp size for O, A and Asia 1 serotypes.As success of any PCR is dependent on the efficiency of primer and template binding, it would be ideal if the primers were designed on the virus sequences native to that particular geographical area.
Multiplex PCR was optimized to amplify the three major serotypes of FMDV A, O and Asia 1 in a single reaction.Primers were designed against the VP1 gene of virus.Initially the primers were optimized individually for each serotype by using gradient temperature PCR.Then all three serotypes were subjected to single multiplex PCR reaction.Annealing temperature for this reaction was optimized at 56.5°C.The amplicon sizes obtained were 386 bp, 240 bp and 232 bp for A, Asia 1 and O serotypes respectively.In mPCR primer to template ratio was optimized.The primer concentration was kept constant at 1.5 μl of 10mM of each forward and reverse primer total 9 μl for three serotypes whereas; template DNA ratio was changed.At template concentration of 1μl, 1.5μl and 2μl non specific products were obtained.The non specific products were obtained because of less quantity of template DNA resulting in primer dimerization.The use of 3μl of cDNA for each serotype and at annealing temperature of 56.5°C has given specific PCR products.At temperatures 53-60°C, specific amplification of the target sequence was found in all the serotypes without much variation in band intensity.The annealing temperature in the study of Alamdari et al., (2006) was optimized at 60 0 C of mPCR.The specific primers were selected from the 2B region of FMDV genome.DNA Fragments of 292, 402 and 732 bp were amplified for the Asia1, O and A serotypes, respectively.There was no difference between amplicon size of serotype O and Asia 1 of FMDV, because of 8 kbp difference between two serotypes that were resolved on 1.5% agarose gel electrophoresis.To resolve this issue amplicon of mPCR were analyzed on 3% PAGE.
The sensitivity of mPCR was determined by making 10-fold serial dilutions of extracted RNA.The serotype A and Asia1 showed the results upto 10 3 dilution with decreasing band intensity whereas; with serotype O results obtained up to 10 4 dilution.This is because sensitivity depends upon length of primer and template DNA concentration.Primers with shorter length were comparatively sensitive than lengthy primers.The specificity of the test in differentiating FMDV infection from other vesicular infections could be validated experimentally.The test specifically detects FMDV infection because primers were designed only against the FMDV genome.The field samples in mPCR, produced a single band of Asia1 serotype of FMDV.Woodbury et al. (1994) analyzed field specimen (Sau 8/88) from Saudi Arabia and reported that O serotype was dominant followed by Asia 1 to some extent.The results were different from present study due to varied geographical distribution of different serotypes.
It is evident from the present study that multiplex PCR is a quick, time saving, cost effective and efficient method to detect different subtypes in a single reaction.It is therefore a helpful tool for the diagnosis of FMDV leading to its effective control and prevention.

Table 1 :
Sequences of used primers