Rep-PCR in Microbial Molecular Identification

Identification and classification of bacteria play an important role in microbial ecology. Many different phenotypic and genotypic methods are currently being used to identify and classify microorganisms. Nucleic acid and PCR-based (genotypic) methods are increasingly being employed and are emerging as reliable, simple, and often inexpensive ways to identify and classify microbes. PCR-based typing methods can scan part of or the entire microbial genome. Creative Biolabs' deep industry expertise coupled with a multi-platform approach delivers tailored high-performance solutions for microbial identification.

Overview of Rep-PCR

The repetitive element sequence-based PCR (rep-PCR) genomic fingerprinting protocols have been applied successfully in a variety of medical, agricultural, industrial, and environmental studies of microbial diversity. In addition to studying diversity, rep-PCR genomic fingerprinting has become an important tool for bacterial identification and classification, and molecular epidemiological studies (diagnostics) of human and plant pathogens. The rep-PCR genomic fingerprinting generated from bacterial isolates can distinguish bacteria at the species, subspecies, and strain levels, and may be the most discriminatory and reproducible genomic fingerprinting method ever used for rapid analysis of multiple populations.

Rep-PCR Procedures

1. Template Preparation for Rep-PCR Genomic Fingerprinting

Several template preparation methods can be used for rep-PCR-mediated genomic fingerprinting. The method chosen depends on the nature of the microorganisms to be analyzed, their receptivity to lysis (releasing DNA), the number of isolates to be analyzed, and the required resolution or available time.

2. Determination and Adjustment of DNA Concentration

When isolated DNA is used for rep-PCR reactions, the concentration should be adjusted to 50 ng/μl (50 μg/ml).

3. Rep-PCR Fingerprinting Protocol

Often a detailed characterization of strains needs to be obtained by applying only one primer. In this case, the BOX primer is generally recommended, since it generates robust fingerprints, and generally yields a highly complex profile of amplified fragments.

  • PCR amplification
  • Agarose gel electrophoresis-mediated separation of rep-PCR amplified genomic fragments

Most rep-PCR genomic fingerprinting applications include ethidium bromide staining for agarose gel electrophoresis, but silver staining for agarose gel and polyacrylamide gel is also commonly used. Normally, an agarose gel is sufficient to separate the rep-PCR generated fragments, although polyacrylamide gels or capillary electrophoresis may also be used for increased precision.

Principle of rep-PCR genomic fingerprinting.Fig.1 Principle of rep-PCR genomic fingerprinting. (Rademaker, 2004)

Advantages of Rep-PCR

A major advantage of the rep-PCR genomic fingerprinting approach is that reproducible fingerprints can be generated not only from purified genomic DNA but also directly from crude lysates, whole cells from liquid cultures or colonies on plates, and infected (plant) tissues, such as root nodules or plant lesions.

Rep-PCR genomic fingerprinting is a very valuable tool in molecular microbial ecology for the identification and classification of microbial isolates. At Creative Biolabs, delivering the best rep-PCR services is just the start; we make sure each client gets our utmost attention and each project benefits from a customized service plan. If you are interested in our rep-PCR services for microbial identification, please contact us for more detail.

Reference

  1. Rademaker, J.L.W.; et al. Characterization of the diversity of ecologically important microbes by rep-PCR genomic fingerprinting. Molecular microbial ecology manual. 2004, 1(2): 611-643.

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