Protocol

Centre : Indian Agricultural Research Institute,New DelhI
Name of the Protocol
Detection of Polyketides producing Bacillus spp. by Multiplex Polymerase Chain Reaction (PCR)
Nature : Novel
Crop / Pathogen : N/A

Inventors

  1. Dr. Dinesh Singh
  2. Dhananjay Kumar Yadav
  3. Garima Chaudhary

Objectives

  1. Screening and detection of Polyketides antibiotic genes producing Bacillus spp. from genomic DNA.
  2. Detection of Polyketides antibiotic genes producing Bacillus spp. direct from soil samples.

Procedure / Summary

A PCR protocol was used to amplify from obtained template in a reaction mixture of 5X taq buffers, 1.5U taq polymerase (Promega), 10 mMdNTPs, 25 mM MgCl2, 1 µl of each forward and reverse primers ( 10 µM) 1 µl of DNA (100 ng) and sterile H20 in final volume of 25 µl. The thermo cycling conditions are initial denaturation 950C for 5 min, followed by 32 amplification cycles of 950C for 30 sec, 520C for 0.45 sec and 720C for 1 min and final extension 720C for 8 min using a BIO-RAD C1000 thermo cycler. The PCR products were resolved by using a 1.5% agarose gel stained with ethidium bromide and photographed using the gel documentation system .To determine the detection threshold of multiplex PCR primer sets of polyketides genes were done by diluting upto 10-3 of genomic DNA (100 ng) of B. amyloliquefaciens ,1.0 µl of aliquots were used as DNA template.Specificity of these primer sets was tested by using DNA template of B. subtilis, B. licheniformis, B. pumulis, B. cereus, Pseudomonas fluorescens, Ralstonia solanacearum, and Xanthomonas campestris pv. campestris along with Bacillus amyloliquefaciens DSBA-11.

Applications

  1. Bacterial DNA is amplified by multiplex PCR to screen Polyketides antibiotic genes producing Bacillus spp. direct from genomic DNA and soil samples.
  2. Multiplex PCR methods exhibit great flexibility in experimental design and in overcoming limiting primer kinetics and fragment competition. A number of uniplex PCR techniques have been adapted to multiplex amplification for detection of antibiotic genes of Bacillus species.