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Standardization of Multiplex Reverse Transcription-polymerase Chain Reaction and Typing of Foot-and-mouth Disease Virus Prevalent in Bangladesh .

M. A. Zinnah
Department of Microbiology and Hygiene, Faculty of Veterinary and Animal Science, Sylhet Agricultural University, Sylhet, Bangladesh.

M. A. Islam
Department of Microbiology and Hygiene, Faculty of Veterinary and Animal Science, Sylhet Agricultural University, Sylhet, Bangladesh.

M. S. R. Khan
Department of Microbiology and Hygiene, Faculty of Veterinary and Animal Science, Sylhet Agricultural University, Sylhet, Bangladesh.

M. H. Haque
Department of Animal Husbandry and Veterinary Science, Rajshahi University, Rajshahi

M. R. Bari
School of Agriculture and Rural Development, Bangladesh Open University, Gazipur-1705, Bangladesh

M. A. Zinnah
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M. T. Hossain
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M. M. Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M. T. Islam
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

Foot-and-mouth disease (FMD) is a devastating viral disease of cattle that causes severe economic losses in terms of loss of production and calf mortality in Bangladesh. Despite of regular vaccination, outbreak of the disease has become a regular event throughout the country every year. Determination of prevailing serotypes of the causal agent foot-and-mouth disease virus (FMDV) is now crucial need for strategic vaccination programme. The present research work was aimed to standardize a multiplex RT-PCR assay typing of foot-and-mouth disease virus serotypes prevalent among cattle population of Bangladesh. Uniplex and multiplex RT-PCRs were successfully developed and standardized using the extracted RNA of reference FMDV (Type A, O and Asia 1) following adjustment of the concentration of the viral RNA of each serotype, volume of reaction mixture and thermal profile. The mPCR was evaluated on 82 field samples (vesicular fluid, tongue epithelium and tissue from inter-digital space) of the years 2007 and 2008. Of the 82 field samples, 56 (68.29%) were found positive for FMDV. The mPCR successfully differentiated single as well as dual serotypes infection. The serotypes A, O and Asia 1 were confirmed in the samples of the year 2007 and only serotype O in samples of the year 2008. Higher detection rate was found in vesicular fluid (100%) followed by tongue epithelium (79.66%). It may be concluded that the MRT-PCR standardized in this study could be used for detection and differentiation of FMDV serotypes using field samples.

Keywords: Foot-and-mouth disease virus, Serotypes, Multiplex RT-PCR

Location: Dhaka, Narshingdi, Rangpur, Comilla and Chittagong districts, Bangladesh

Start Date: 00-00-2007

End Date: 00-00-2008

Program Area: Animal Health and Management

Commodity: Diseases

Objectives:

To identify the standardization of multiplex reverse transcription-polymerase chain reaction and typing of foot-and-mouth disease virus prevalent in Bangladesh .

Methodology:

The three different serotypes (A, O, Asia 1) of reference FMDV were obtained from the repository of the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh. A total of 82 samples (vesicular fluid, tongue epithelium and tissue from inter-digital space) were collected from suspected foot-and-mouth disease (FMD) affected cattle of different outbreak areas of five districts. The samples were kept at -86⁰C until used. The field samples (tongue epithelium, tissue from inter-digital space) were homogenized with sea sand using mortar and pestle and 20% suspensions were prepared by adding sterile phosphate buffered saline. The suspension was then centrifuged at 3000 rpm for 10 min maintaining 40C and supernatant was collected for extraction of viral RNA. The genomic viral RNA was extracted from field samples by using QIAamp RNA mini kit (Hilden, Germany) according to the manufacturer’s instructions. Initially, 4 μl of template RNA and 100 (0.5μl) pmols of RT-primer (RH) were heated at 70⁰C for 10 min followed by snap cooling on ice. Then 15.5 μl reaction mixture containing 4 μl 5X RT buffer, 2 μl 10 mM dNTPs, 1 μl prime RNase inhibitors, 0.2 μl AMV-RT and 8.3 μl of RNase free water was added to the PCR tube containing RNA of FMDV. The RT was carried out at 420C for 1 hour in a thermocycler followed by heating at 95⁰C for 5 min. RT products were cooled on ice and stored at -20⁰C until use. For uniplex PCR of each serotype of FMDV, the basic PCR reaction mixture (50 μl) contained 2 μl of RT product, 5 μl of 10X PCR buffer (LA buffer, Takara, Japan), 2 μl of 25 mM MgCl2, 2 μl of 10 mM dNTP, 100 pmols (0.8 μl) each of virus specific and serotype specific primers (Table 1), 0.2 μl of LA Taq DNA polymerase, 38 μl of RNase free water. It was subjected to following thermal cyclic conditions: one cycle at 95⁰C for 15 min, 30 cycles each at 95⁰C for 30 s, 55⁰C for 30 s, and 72⁰C for 1 min followed by one cycle at 72⁰C for 10 min. For multiplex PCR, initially the reaction mixture and cyclic conditions were same as uniplex PCR. Standardization of multiplex PCR was done by varying reaction components and cyclic conditions one at a time. Multiplex PCR was performed on all the serotypes at unit incremental annealing temperatures from 560C to 700C and then at 72⁰C to 74⁰C. Reaction components and rest of the cyclic conditions were kept constant. However, annealing temperature of 58⁰C was selected to carry out mPCR on the basis of melting temperature of each primer of each serotype. At the selected annealing temperature of 58⁰C, mPCR was carried out at 2 μl of 25 mM MgCl2 using 10 mM dNTP. At a constant MgCl2 concentration, the influence of 2 μl of 10 mM dNTP was evaluated. A 0.2 μl of LA Taq DNA polymerase was used in mPCR for all the serotypes. Different concentration (10 to 100 pmol) of each primer for each serotype was used and on the basis of band intensity, the final concentration (100 pmol) of each primer was selected for mPCR. After determining the optimum concentration of reaction mixture and annealing temperature of the primers, the cyclic condition, such as denaturation, annealing and extension time and also the number of cycles were adjusted for optimal performance. Based on the performance, the following cyclic conditions were selected for optimal performance: one cycle of 95ºC for 15 min, 30 cycles of 95ºC for 30 s, 58ºC for 30 s, 72ºC for 60 s and one cycle of 72ºC for 10 min. The PCR products were electrophoresed at 100V for 30 min in TAE buffer on 2% agarose gel containing ethidium bromide (0.6 mg/ml). DNA molecular weight marker type 100 bp DNA ladder was included to identify the size of the PCR products, using a GelMate 2000 (Toyobo) and UV-transilluminator (UVP Life Sciences).

Source of Information: Bangl. J. Vet. Med. (2010). 8 (2) : 149 – 155

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

From the results of typing of FMDV in field samples by using MRT-PCR, it can be seen that MRT-PCR worked more efficiently to detect mixed infection with serotypes A and O. However, it would be worthwhile to have a sample to be tested from cases of multiple infections with all the three serotypes. The detection rate of FMDV by MRT-PCR in different types of samples varied from 10% to 100%. This variation might be due to the concentration of viruses in the samples. The exposure of specimens to higher environmental temperatures, incorrect pH or putrefaction of the specimen might have contributed to virus degradation and lead to lowered virus or antigen concentrations from which sufficient intact RNA may or may not be extracted for the RT-PCR to function allowing amplification of the specific region of the viral genome. Besides, tissues of interdigital space of cattle are frequently exposed to soil, mud, naphthalene and oil of turpentine (used as fly repellent) and so on which possibly destroyed the FMDV to greater extent. It may be concluded that the MRT-PCR standardized in this study could be used for detection and differentiation of FMDV serotypes using field samples. However, it would be ideal to determine the sequence of the PCR products obtained by this MRT-PCR for more confirmation and to evaluate this test on more FMD suspected samples from different parts of Bangladesh.

Knowledge Management: Journal