Development of polymerase chain reaction-based assays for bacterial gene detection

doi:10.1016/S0167-7012(00)00160-3

Journal of Microbiological Methods

Volume 41, Issue 3, August 2000, Pages 201-209

https://doi.org/10.1016/S0167-7012(00)00160-3 Get rights and content

Abstract

Polymerase chain reaction-based assays provide rapid, simple, and sensitive detection of bacterial genes, but are not without their drawbacks. This review summarizes the principal advantages and disadvantages of PCR-based bacterial gene detection, provides guidelines for the development and validation of new PCR assays, and describes potential pitfalls that may be encountered and how these can be avoided.

Section snippets

Diagnostic PCR in bacteriological research

The polymerase chain reaction (PCR) has been a great boon to bacteriological research. Its many uses include the detection of specific DNA or RNA sequences (Johnson and Brown, 1996, Persing, 1996), DNA sequencing, amplification fingerprinting (Welsh and McClelland, 1990, Caetano-Anolles, 1993, Versalovic and Schneid, 1994), detection and identification of non-culturable microorganisms (Fredricks and Relman, 1999), and site-directed mutagenesis (Link and Phillips, 1997). The most familiar of

‘Is PCR really the best approach?’

However, before developing a new diagnostic PCR assay or adopting an established one, it behooves the investigator to consider the potential disadvantages of PCR. Reagents and disposable materials (e.g., special PCR tubes and pipettor tips) are fairly expensive, particularly in comparison with those required for many traditional phenotypic tests or probe hybridization, which today can be done without radioactivity or the use of toxic chemicals such as phenol (Fredricks and Relman, 1999).

Sequence

Fundamental to the development of PCR-based gene detection assays is the knowledge of the nucleotide sequence of the gene of interest. The appropriate DNA sequences usually can be located by searching the GenBank databases (http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html) with keywords that include the gene name and species. In many instances, sequence will be available for multiple versions of the gene from different strains, in which case it is advisable to retrieve and compare the

Primer design

Many computer applications are available to assist with initial primer selection, including Primer 3 (Rozen and Skaletsky, 1998), Primer Designer (version 4, 1994, Scientific and Educational Software, Durham, NC: see http://www.scied.com/ses_pd4.htm), and others (see http://biochem.boehringer-mannheim.com/prod_inf/manuals/pcr_man/Chapter02/CHAP01-Seite12.htm). Primer design programs typically allow the user to specify various parameters, after which the computer searches the designated target

Assay development and optmization

The single best candidate forward and reverse primers are next synthesized. A post-synthesis ‘clean up’ step is desirable to remove residual salts which might intefere with the amplification reaction (Cave and Bingen, 1994). This can be accomplished by sepharose column purification, which is often included in the purchase price of commercially-synthesized primers. HPLC purification, which eliminates mutant primers that might interfere with structural analyses, is available for an extra charge.

Assay validation

Although the demonstration that a newly-designed primer pair can generate an amplicon of the expected size from positive control DNA and fail to react with a negative control sample is encouraging, this really is only the first step in the validation of a new PCR assay. It is important to more extensively confirm both the specificity and the sensitivity of the assay (Fredricks and Relman, 1999). Specificity can be further confirmed by demonstrating (1) that the amplified band actually

Multiplexing

An increasingly popular maneuver that increases the efficiency of diagnostic PCR (although possibly interfering with sensitivity in some circumstances) is to combine multiple primers in one PCR reaction so as to simultaneously amplify multiple different gene regions, a process sometimes termed ‘multiply primed’ or ‘multiplex’ PCR (Le Bouguenec and Archambaud, 1992, Yamamoto and Terai, 1995, Johnson and Brown, 1996, Karkkainen and Kauppinin, 1998, Mitsumori and Terai, 1998, Johnson and Stell,

Summary

Diagnostic PCR is a powerful tool for bacteriological research. Although in some circumstances alternative methods may be preferable, PCR assays do offer many potential advantages over traditional methodologies. New assays can be developed readily even by investigators with only a modicum of experience with PCR, so long as established principles are observed. Scrupulous assay validation and careful attention to quality control during assay use are essential.

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Invited reviewDevelopment of polymerase chain reaction-based assays for bacterial gene detection

Abstract

Section snippets

Diagnostic PCR in bacteriological research

‘Is PCR really the best approach?’

Sequence

Primer design

Assay development and optmization

Assay validation

Multiplexing

Summary

Res. Microbiol.

Trends Biochem. Sci.

Methods Enzymol.

J. Microbiol. Methods

FEMS Immunol. Med. Microbiol.

FEMS Immunol. Med. Microbiol.

Amplifying DNA with arbitrary oligonucleotide primers

PCR Method. Appl.

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications

Nucleic Acids Res.

Pcr Primer: A Laboratory Manual

Touchdown PCR to circumvent spurious priming during gene amplification

Nucleic Acids Res.

Application of polymerase chain reaction to the diagnosis of infectious diseases

Clin. Infect. Dis.

Method to improve reliability of random-amplified polymorphic DNA markers

Biotechniques

Multiplex PCR: critical parameters and step-by-step protocol

Biotechniques

Optimization of PCRs

A novel multiply-primed polymerase chain reaction assay for identification of variant papG genes encoding the Gal(αl-4)Gal-binding PapG adhesins of Escherichia coli

J. Infect. Dis.

Colonization with and acquisition of uropathogenic Escherichia coli strains as revealed by polymerase chain reaction-based detection

J. Infect. Dis.

papG alleles of Escherichia coli strains causing first episode or recurrent acute cystitis in adult women

J. Infect. Dis.

Invited review
Development of polymerase chain reaction-based assays for bacterial gene detection