PCR Amplification-Based Microbial Typing

2013

Identification and classification of the microorganisms are of utmost importance in the field of environmental, industrial, medical and agricultural microbiology, and microbial ecology. Traditional phenotype-based methods encounter many challenges and shortcomings which limit their usability. Molecular methods offer better solutions in identifying and characterizing microorganisms. Several DNA fingerprinting methods have been developed and are in use already. In principle, most of these methods are based on PCR and restriction site analysis. Some of these methods are still not economic in use and require huge set-up cost. Continuous research is going on around the world to improve the methodology and applicability of these methods as well as to make them economic in use.

Analytical Chemistry, 2005

Bloodstream infections are an important cause of serious morbidity and mortality. Rapid detection and identification of specific pathogens from blood or other clinical specimens could improve the rational use of antimicrobial therapy in clinical medicine and have a great impact on the outcome of patients with systemic infections. Polymerase chain reaction using generic primers was used to amplify genomic DNA of different bacterial strains. The identification was accomplished by measuring the molecular masses of the PCR products using ion-pair reversedphase high-performance liquid chromatography hyphenated to electrospray ionization mass spectrometry. DNA from 10 bacterial species was amplified by PCR, and the resulting amplification products were analyzed. In all cases, the measured molecular masses of the PCR products matched the theoretical value for the species-specific DNA sequence. However, three pairs of bacteria could not be distinguished since the theoretical difference in amplicon molecular mass was <1.0 Da (different sequence, same base composition of amplicon). Determination of intra-and interday mass reproducibility resulted in relative standard deviations of 0.0030 and 0.018%, respectively. The limit of detection of the presented method was shown to be 0.5 genome equivalents/PCR. The specificity of the method in a human background was successfully tested by amplifying and analyzing 1000-10000 genome equivalents of Staphylococcus aureus spiked into human plasma.

Applied and Environmental Microbiology, 2005

The proposed technique is based on the digestion of genomic DNA with the restriction endonuclease Sau3AI and subsequent amplification with primers whose core sequence is based on the Sau3AI recognition site. The method was tested on strains of lactic acid bacteria but could be proposed for virtually any culturable organism from which DNA can be extracted.

2006

Microbial Genomics in Sustainable Agroecosystems, 2019

The advent in molecular biology techniques has enabled, to a great extent, numerous identification and detection techniques of microorganisms by amplifying specific conserved DNA sequences. From time-to-time, the tremendous modifications have been employed in search of a rapid and inexpensive microbial identification method and now a whole genome to be sequenced is possible. In this chapter, we have described the different molecular identification techniques including 16S/18S rRNA, ITS and whole-genome sequencing as well as genotyping techniques such as pulse field gel electrophoresis, AFLP, RAPD, RFLP, ribotyping, BOX, ERIC, rep-PCR and multi-locus sequence typing with their basic principle, procedure, strengths and weaknesses.

Electrophoresis, 1998

The polymerase chain reaction (PCR) is a powerful molecular biology tool which can be used for the identification of species and strains of diverse microorganisms. By aimed amplification of characteristic genes (i.e., genes encoding ribosomal RNA molecules) and subsequent genetic analysis of amplified fragments, information on microbiological systematics and phylogeny can be obtained in a fast and efficient manner. Similar types of gene identification can be used to verify or detect genes responsible for phenotypic characteristics, whereas modified forms of the PCR enable whole genome searches for genetic polymorphisms among strains of a given species. In medical sciences, both strategies, gene and genome variability analysis by PCR, have an increasing impact on the study of the spread of especially those microbes that are multiply resistant to clinically used antibiotics. In this communication we will exemplify the usefulness of PCR-mediated typing of microorganisms from a clinical perspective while focusing on gene- versus genome-scanning. Special emphasis will be placed on analysis of the dissemination and characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains and bacterial factors providing resistance to penicillin and other β-lactam antibiotics. Technical limitations and possibilities for improvement will be discussed.

Biotechniques, 2000

Electrophoresis, 1994

Microbiology-sgm, 1996

We investigated the usefulness of a novel DNA fingerprinting technique, AFLP, which is based on the selective amplification of genomic restriction fragments by PCR, to differentiate bacterial strains at the subgeneric level. In total, 147 bacterial strains were subjected to AFLP fingerprinting : 36 Xanthomonas strains, including 23 pathovars of Xanthomnas axonopodis and six pathovars of Xanthomonas vasicola, one strain of Stenotrophomonas, 90 genotypically characterized strains comprising all 14 hybridization groups currently described in the genus Aemmonas, and four strains of each of the genera Clostridium, Bacillus, Acinetobacter, Pseudomonas and Vibrio. Depending on the genus, total genomic DNA of each bacterium was digested with a particular combination of two restriction endonucleases and the resulting fragments were ligated to restriction halfsite-specif ic adaptors. These adaptors served as primer-binding sites allowing the fragments to be amplified by selective PCR primers that extend beyond the adaptor and restriction site sequences. Following electrophoretic separation on 5 O h (w/v) polyacrylamide/83 M urea, amplified products could be visualized by autoradiography because one of the selective primers was radioactively labelled. The resulting banding patterns, containing approximately 3&50 visualized PCR products in the size range 80-550 bp, were captured by a highresolution densitoscanner and further processed for computer-assisted analysis to determine band-based similarity coefficients. This study reveals extensive evidence for the applicability of AFLP in bacterial taxonomy through comparison of the newly obtained data with results previously obtained by well-established genotypic and chemotaxonomic methods such as DNA-DNA hybridization and cellular fatty acid analysis. In addition, this study clearly demonstrates the superior discriminative power of AFLP towards the differentiation of highly related bacterial strains that belong to the same species or even biovar (i.e. to characterize strains at the infrasubspecif ic level), highlighting the potential of this novel fingerprinting method in epidemiological and evolutionary studies. 2 KeyGene nv, Wageningen, Keywords : DNA fingerprinting, PCR, selective amplification, AFLP 0002-0556 0 1996 SGM C. acetobutylicum ATCC 824T C. acetobutylicuni DSM 792T C. acetobutylicum DSM 1731 C. beijerinckii NCIMB 8052* B. cereus LMG 8221 B. cereus LMG 6923T B. thuringiensis LMG 12267 B. larvae LMG 9820T Ac. baumannii LMG 1041T Ac. baumannii LMG 994 Ac. baumannii LMG 10524 Ac. lwofJii LMG 1029T Ac. baumannii LMG 1041T Ac. baumannii LMG 10524 Ac. baumannii LMG 994 Ac. LwofJii LMG 1029T V. anguillarunr VIB I 1 3 V. anguillarum VIB 156 ! V. anguillarum VIB 94 V. tubiashii V1B 309 6 V. anguillarum VIB 113 1 V. anguillarum VIB 94 I V. anguillarum VIB 156 I V. tubiashii VIB 309 P. aeruginosa LMG 5033 P. aeruginosa LMG 6395 P. aeruginosa LMG 5827