Evaluation of a protective antigen gene based SYBR Green I real time PCR for detection of Bacillus anthracis in field samples


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Authors

  • R KHANDIA
  • B PATTNAIK
  • K RAJUKUMAR
  • A K PATERIYA
  • H V MURUGKAR
  • S BHATIA
  • A PRAKASH
  • H K PRADHAN

Keywords:

Bacillus anthracis, Sequence variation, SYBR Green PCR, Tm variation

Abstract

In the present study, 27 field materials from different sources were tested in SYBR Green I dye based Real Time PCR optimized using WHO recommended conventional PCR primers targeting Bacillus anthracis protective antigen PA) gene of pX01 plasmid. The assay had detection limit of 0.000002 pg in terms of target DNA quantity and up to 4 copies of pX01 plasmid per reaction, in terms of copy number. Melt curve analysis of PCR products of field samples showed deviation up to 3.2OC in melting temperature from standard anthrax strain suggestive of possible mutation mutations in PA gene. Targeted region of PA (596 bp) encompasses domain 2, involved in its binding with lethal factor LF) and/or edema factor (EF) and mutation in this region may alter the efficacy of PA to traffic toxins inside the cells and results in differed level of virulence of pathogen. The test is useful not only for detection but may be suggestive of mutation also.

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Author Biographies

  • R KHANDIA
    Research Associate, High Security Animal Disease Laboratory, IVRI, Anand Nagar, Bhopal, Madhya Pradesh 462021 India
  • B PATTNAIK
    Project Director, Project Directorate on Foot and Mouth Disease, Mukteswar - Kumaon, Nainital, High Security Animal Disease Laboratory, IVRI, Anand Nagar, Bhopal, Madhya Pradesh 462021 India



  • K RAJUKUMAR
    Scientist (SS),  High Security Animal Disease Laboratory, IVRI, Anand Nagar, Bhopal, Madhya Pradesh 462021 India




  • A K PATERIYA
    Scientist,  High Security Animal Disease Laboratory, IVRI, Anand Nagar, Bhopal, Madhya Pradesh 462021 India



  • H V MURUGKAR
    Senior Scientist,  High Security Animal Disease Laboratory, IVRI, Anand Nagar, Bhopal, Madhya Pradesh 462021 India



  • S BHATIA
    Senior Scientist,  High Security Animal Disease Laboratory, IVRI, Anand Nagar, Bhopal, Madhya Pradesh 462021 India

  • A PRAKASH
    Professor, Department of Biotechnology, Barkatullah University, Bhopal, High Security Animal Disease Laboratory, IVRI, Anand Nagar, Bhopal, Madhya Pradesh 462021 India



  • H K PRADHAN
    Avian Influenza National consultant, Indian
    Office of WHO consultant, Bhartiya Kala Kendra, 1-Copernicus marg, New Delhi,

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How to Cite

KHANDIA, R., PATTNAIK, B., RAJUKUMAR, K., PATERIYA, A. K., MURUGKAR, H. V., BHATIA, S., PRAKASH, A., & PRADHAN, H. K. (2013). Evaluation of a protective antigen gene based SYBR Green I real time PCR for detection of Bacillus anthracis in field samples. The Indian Journal of Animal Sciences, 83(2). https://epubs.icar.org.in/index.php/IJAnS/article/view/27237