The diversilab system for dna fingerprinting and analysis is a powerful tool for tracking the spread and source of microbial infection, contamination, or epidemics natural and manmade. Antimicrobial resistance and genomic reppcr fingerprints. Reppcr and rapdpcr fingerprinting of aspergillus terreus. On the basis of an 80% similarity threshold, rep pcr consistently clustered like species and this set of isolates, along with five atcc reference strains, was used to create a dna fingerprint library with the diversilab software. As already observed with mlst, the concordance of dl with pfge was low for all three ccs wallace coefficient rep pcr. Predicting salmonella enterica serotypes by repetitive. Rep pcr based dendrogram and virtualgel image fingerprints for 71 isolates of different salmonella serovars following typing using the diversilab system with the salmonella kit. Pdf yeast identification by sequencing, biochemical kits. Diversilab, which is one of those methods, is a commercial repetitivesequencebased pcr reppcr typing system that amplifies strainspecific noncoding repetitive sequences. Based on patented rep pcr technology, microbial isolates can quickly and accurately be distinguished at the subspecies and strain level. Comparison of a commercially available repetitiveelement. Yeast identification by sequencing, biochemical kits. The bacterial kit is designed to be used on all gram positive and gram negative bacteria, however it cannot. Using pcr technology, diversilab enables you to quickly differentiate microbial isolates at a subspecies and strain level in case of contamination.
The performance of repetitivesequencebased pcr reppcr using the diversilab system for identification of dermatophytes commonly isolated in a clinical laboratory was assessed by comparing results to those of conventional tests colony morphology, microscopic examination of slide cultures, and, for suspected trichophyton species, use of additional media. All salmonella isolates were capable of being typed with the diversilab system and salmonella dna fingerprinting kit. Th e analysis utilized the the rep pcr technology and rep pcr primers are covered by u. Conventional morphology versus pcr sequencing, rep.
Six and eight distinct fingerprinting types were found with primer mixes for. Diversilab was compared to pcr ribotyping of the 16s23s rna intergenic spacer of clostridium difficile cd as the gold standard method for cd typing. Diversilab fingerprinting kits previously called bacterial bar codes, a type of repetitiveelement pcr rep pcr, is a rapid, semiautomated, pcr based, commercial typing system that can be easily performed by bench technologists with some molecular experience. We describe the adaptations made to automate reppcr and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. Ninetyfive clinical isolates, identified by conventional methods, and five atcc strains were tested. For analysis, diversilab software utilized the pearson correlation coef.
The rep pcr results apparently suggested a higher level of genetic variability, and both the dendrogram and the similarity matrix generated by the diversilab software fig. The diversilab system is an automated platform that uses reppcr technology to provide standardized, reproducible dna fingerprinting and analysis of bacterial and fungal samples for complete isolate characterization. Yeast identification by sequencing, biochemical kits, malditof ms and rep pcr dna fingerprinting. Diversilab system dl biomerieux, marcy etoile, france was introduced a semiautomated reppcr system. The automated the automated detection and standard algorithms for data analysis adds consistency to this molecular. Interlaboratory reproducibility of diversilab rep pcr typing and clustering of acinetobacter baumannii isolates article pdf available in journal of medical microbiology 61pt 1. The diversilab system exploits the discriminatory power of reppcr. Use of the diversilab semiautomated repetitivesequence. Comparison of a commercially available repetitiveelement pcr system diversilab with pcr ribotyping for typing of clostridium difficile strains. Dna was extracted and genomic dna was amplified using the diversilab mycobacterium typing kit. Similarly, vogel and colleagues 2007 used automated rep. The diversilab system generated rep pcr dna fingerprints for each of the 26 fusarium isolates, and the similarity comparisons are shown as a dendrogram fig. Dl bacteria kit 24 test dl reagents and consumables dl.
Use of the diversilab repetitive sequencebased pcr system for genotyping and identification of archaea article in journal of microbiological methods 732. The diversilab reppcr assay is a rapid, semiautomated method based on pcr amplification of specific regions between noncoding repetitive sequences in the bacterial genome. Routine molecular typing using rep pcr would not only increase annual material costs by 32%, but it would also require an additional 0. On the basis of an 80 % similarity threshold, rep pcr consistently clustered like species and this set of isolates, along with five atcc reference strains, was used to create a dna fingerprint library with the diversilab software. The diversilab system spectral genomics, houston, tx is a rapid, technically simple method that uses repetitivesequencebased pcr reppcr to determine relatedness of many organisms and to identify aspergillus and candida to the species level 2, 7. Repetitivesequencebased pcr reppcr using the diversilab system was investigated for identification of aspergillus.
The costs of routinely performing pcr ribotyping with analysis of the pcr products using a diversilab chip compared to automated rep pcr diversilab is outlined in table 3. For analysis, diversilab software utilized the pearson correlation coefficient and the unweighted pairgroup methods of averages upgma to automatically compare the rep pcr profiles and create corresponding dendrograms healy et al. Use of the diversilab repetitive sequencebased pcr system. The resulting reppcrbased dna fingerprints were uploaded to and analysed using webbased diversilab software through pearsons correlation coefficient.
The dna amplicons were detected, and analyses were performed with the diversilab system and accompanying software v. The diversilab salmonella kit was used for reppcr amplification of intergenic repetitive elements in the genomic dna. Strain typing is suited to track the source of an exact contaminant. Pfge was performed on 31 isolates representative of the most relevant dlrpcs clustered within the 3 major mlst ccs cc17, cc23 and cc1. Comparison of an automated repetitive sequencebased pcr. Pearsons correlation coefficient was used to create a pairwise percentage similarity. Microbial dna typing by automated repetitivesequence. Figure figure1 1 shows rep pcr profiles of the aspergillus isolates. Molecular fingerprinting of candida and aspergillus. These kits contain the reagents and buffers needed to set up pcr reactions using reppcr primers, starting from extracted genomic dna from microbial isolates. The diversilab system biomerieux is an automated dna fingerprinting system based on the reppcr electrophoresis technique.
Recently, reppcr has been commercially adapted to an automated format known as the diversilab system to provide a reliable pcrbased typing system for clinical laboratories. Molecular fingerprinting of clinical candida isolates. Species identification and strain differentiation of. Mitis salivarius with bacitracin msb, golds is a selective media commonly used for the isolation of streptococcus mutans sm, an organism associated with dental caries. Diversilab rep pcr used two different primer mixes for typing aspergillus and other moulds which resulted in all a. The genomic profile, determined by diversilab system rep pcr based technique, was compared with the p. Using a commercial diversilab semiautomated repetitive. These kits contain the reagents and buffers needed to set up pcr reactions using rep pcr primers, starting from extracted genomic dna from microbial isolates. Conventional morphology versus pcr sequencing, rep pcr, and malditofms for identi. The diversilab dna fingerprinting kits are designed to generate reppcr dna fingerprints from purecultured microbial samples. Comparative evaluation of an automated repetitivesequence. It has more advantages than reppcr, such as higher reproducibility, standardization, and archiving fingerprinting patterns and reporting 3,1012.
The amplified fragments of various sizes and fluorescence intensities were separated and detected using a microfluidics chip with an agilent 2100 bioanalyzer. This media is currently being used in a largescale epidemiological study to genotype sm using repetitive extragenic palindromic pcr rep pcr. Detection and analysis of the rep pcr products were performed using the diversilab software version 3. Based on patented reppcr technology, microbial isolates can quickly and. The automated diversilab system provides high performance genotypic characterization of bacteria, yeast and mold. The diversilab system, which includes microfluidicsbased detection, reagent kits, and software for data processing and analysis, is an automated method using repetitive sequencebased pcr reppcr for microbial strain typing. The system is particularly useful for bacterial genotyping and is used for source tracking of hospital and community infection, contamination and epidemiological surveillance.
Laurent, quebec, canada utilizing a 2100 bioanalyzer agilent technologies, santa clara, ca operating the diversilab v1. Pcr the diversilab system and reported that cattle and wildlife were the main sources of e. Use of the diversilab system for species and strain. Diversilab software, which has unlimited, passwordprotected access from any computer with an internet connection, generates userfriendly reports that can be customized for the testing laboratory. Molecular characterization of multidrugresistant klebsiella pneumoniae isolates xianghua hou1, xiuyu song2, xiaobo ma2, shiyang zhang3,4, jiaqin zhang2,3 1department of nephrology, the first affiliated hospital of xiamen university, xiamen, china. The dendrograms were generated automatically using the diversilab software and pairwise groupings with the aspergillus dna fingerprint patterns. Osf repetitive extragenic palindromic pcr reppcr of. The amplified fragments were separated and detected using a microfluidics chip with agilent 2100. Use of the diversilab semiautomated repetitivesequencebased polymerase chain reaction for epidemiologic analysis on acinetobacter baumannii isolates in different italian hospitals.
It can perform same day strain typing for real time investigations. Evaluation of automated repetitivesequencebased pcr. Alternative versus gold standard method for the typing of. Repetitivesequencebased pcr using the diversilab system.
After pcr amplification of genomic dna regions between noncoding intergenic repetitive elements, amplified fragments are separated by microfluidic electrophoresis using agilent technology. Interlaboratory reproducibility of diversilab reppcr. Clinical evaluation of the diversilab microbial typing. Automated discrimination of mrsa and mssa using rep pcr. Repetitivesequencepcrbased dna fingerprinting using the. Around 40% of the isolates expressed serotypes o6 20. The reppcr technology and reppcr primers are covered by u.