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Comparison of Genes as Target for Molecular Diagnosis of Peste Des Petits Ruminants in Goats

A. R. Bhuiyan
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. M. Rahman
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

J. A. Begum
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. R. Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Peste des petits ruminants (PPR) are an acute viral disease of sheep and goats. Rapid and accurate diagnosis is essential for successful control. Reverse transcriptase polymerase chain reaction (RT-PCR), a molecular diagnostic test based on amplification of the gene target is more sensitive than other tests. The study was to find an efficient primer set and structural gene, which would be more specific and sensitive for detecting PPR virus (PPRV) in field samples. Six primer sets for six structural genes of PPR were used. Primer against NP gene (np3/np4) was specific and sensitive. To ensure efficient amplification and detection of viruses in field samples, more than one set of primers should be used and F and N gene specific primers were the most suitable.

Keywords: Efficient primer set, Gene, PPRV, Goats.

Location:

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Goat

Objectives:

The present study was undertaken to find an efficient primer set and gene, which would be specific and sensitive for detecting PPRV in field samples.

Methodology:

Samples: A total of 10 field samples were collected from dead PPR suspects. Mesenteric and bronchial lymph nodes and lung tissues were collected in sterile Falcon tubes. Pooled tissues were macerated using PBS as 20% (w/v) suspension, centrifuged at 800g for 10 minutes. The supernatant was collected in fresh sterile Falcon tubes, gentamycin was added 500 μg/mL and stored at -20°C. Freeze-dried live PPR vaccine from Livestock Research Institute (LRI), Department of Livestock Services and a previously identified isolate were used as the positive controls. When needed RNA was extracted from the same homogenate. PCR: RNA was isolated from the tissue suspension and vaccine virus using RNeasy Kit (Qiagen, Germany) as recommended by the manufacturer. Initially standardized RTPCR specific for F and N genes were used individually to detect PPRV from each suspect. Samples found positive with F and N gene specific primers were used for further RT-PCR using P, M, H and L gene specific primers. In all cases the reaction was set up as 5xQiagen One Step RT-PCR buffer 5 μL, dNTP Mix (containing 10 mM of each dNTP) 1 μL, Primer forward (100 pmol/μL) 0.5 μL, Primer reverse (100 pmol/μL) 0.5 μL, Qiagen One Step RT-PCR Enzyme Mix 1.0 μL, template RNA 5 μL, total reaction volume 25 μL. First the RNA was reverse transcribed by incubating at 50°C for 30 minutes, then the PCR started with an initial denaturation and activation of Taq polymerase at 95ºC for 15 minutes. Annealing, elongation and final extension steps were standardized: The expected band was visualized in 1.5% agarose gel stained with ethidium bromide. The result was viewed on the monitor; the image was printed and saved electronically. Determination of sensitivity of primer: RT-PCR was done further using diluted RNA and primer(s) that detected PPR virus in more field samples. Extracted RNA was quantified, and 10-fold dilution was made (Undiluted, 10-1, 10-2, 10-3, 10-4, 10-5). One step RT-PCR was performed for each dilution. Judgment was made after electrophoresis on 1.5% agarose. The highest dilution that failed to amplify was considered as detection limit. Primers that Determination of sensitivity of primer RT-PCR was done further using diluted RNA and primer(s) that detected PPR virus in more field samples. Extracted RNA was quantified, and 10-fold dilution was made (Undiluted, 10-1, 10-2, 10-3, 10-4, 10-5). One step RT-PCR was performed for each dilution. Judgment was made after electrophoresis on 1.5% agarose. The highest dilution that failed to amplify was considered as detection limit. Primers that successfully amplified at highest dilution without background reaction were considered more sensitive.

Source of Information: Bangl. vet. 2012. Vol. 29, No. 2,56 - 62

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Primer against NP gene (np3/np4) was specific and sensitive for the detection of PPRV from field samples but for efficient amplification and detection of viruses in field samples, it is desirable to have more than one set of primers for diagnosis; and F and N genes are the most suitable.

Knowledge Management: Journal