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Novel Multiplex-pcr for Rapid Detection of Bacillus Anthracis Spores Present in Soils of Sirajganj District in Bangladesh

KHMNH Nazir*
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

SMZH Chowdhury
Bangladesh Agricultural Research Council, Farmgate, Dhaka, Bangladesh

J Hassan
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

MB Rahman
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Bacillus anthracis spores were isolated and detected from soil samples (n=72) using multiplex-PCR method. The bacteria were isolated and primarily identified as Bacillus anthracis using selective Polymyxin B - Lysozyme - EDTA - Thallous acetate agar. A multiplex-PCR method targeting three genes; rpoB of genome, pag of pX01 and cap of pX02 was done to confirm the isolated bacteria. Among 72 soil samples, the viable B. anthracis spores could be extracted from 14 (19.44%) samples. However, both pX01 and pX02 plasmids were harbored in 5 (6.94%) isolates. On the other hand, pX01 and pX02 was present in 8 (57.14%) and 11 (78.57%) isolates, respectively. This two-step-method was found to be easy, accurate and rapid in identification of B. anthracis spores from soil samples and to identify the toxigenic plasmids present in this bacterium.

Keywords: Anthrax, Bacillus anthracis, Spore, Multiplex-PCR, Soil

Location: Sirajganj district, Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

In this study, taking an advantage of multiplex-PCR method, we established a two-step-method for an easy, precise and rapid identification of B. anthracis spore from soil samples targeting the presence of virulent plasmids (pX01 and/or pX02).

Methodology:

To extract the spores, approximately 400-gm of surface soil samples (n=72) were collected from Sirajganj district, Bangladesh (Ahsan et al., 2013). Using the selective Polymyxin B - Lysozyme - EDTA - Thallous acetate (PLET) agar (SigmaAldrich, Switzerland), the viable spores were extracted (Dragon and Rennie, 2001; Ahsan et al., 2013). From the B. anthracis positive plates, colonies (one colony from each) were taken and cultured overnight at 37o C in liquid culture media. Total DNA was extracted following the procedures of Jula et al. (2007) with some modifications. Combination of three primers targeting portions of rpoB gene (359- bp, specific for Bacillus genus; 208-bp, specific for Bacillus anthracis) present in B. anthracis chromosome were used in the multiplex-PCR (Ko et al., 2003). In addition, two pairs of primer sets were designed targeting portions of pag gene of pX01 (287-bp) and cap gene of pX02 (164-bp) by analyzing the nucleotide sequences of these two plasmids collected from GenBank database. The oligonucleotide primers (Table 1) were purchased from the Cosmo Genetech Co., Ltd., South Korea. The multiplex-PCR was performed according to the procedure described by Ko et al. (2003) with some modifications. In brief, the PCR assay was performed in a total reaction volume of 25 µl consisting of ca. 50 ng template DNA, 2.5 µl of 10 x PCR buffer, 0.2 mM dNTPs mix (SolGent Co., Ltd.). Thirty five cycles of amplification were performed in Thermal Cycler (AB Applied Biosystem) following initial denaturation at 95°C for 2 min. Each cycle consisted of a denaturation step at 95°C for 30 s, an annealing step at 45°C for 30 s, and an extension step at 72°C for 30 s, with a final extension at 72°C for 1 min The PCR products were analyzed by electrophoresis on 2% (wt/vol) agarose gels stained with ethidium bromide (0.5 mg/ml). A previously isolated B. subtilis isolate (unpublished) was used as a negative control. The purified PCR fragments generated by using specific primers targeting pX01 and pX02 were validated by sequencing using automatic DNA sequencer (ABI 3730XL; Applied Biosystem). The PCR reaction and condition were same as described above.

Source of Information: Progressive Agriculture 26:67-70, 2015 ISSN: 1017 - 8139

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The newly established two-step-method was found sensitive to identify virulent strains of B. anthracis. The findings of this study can be used as an easy, precise and rapid screening of B. anthracis from soil and other environmental samples.

Knowledge Management: Journal