Use of the Agilent 2100 Bioanalyzer for Rapid and Reproducible Molecular Typing of Streptococcus pneumoniae

Abstract

Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.Streptococcus pneumoniae is an important human pathogen with a clonal population structure (4). Molecular typing has become increasingly important in order to understand the evolution of this pathogen and to trace clones with special traits such as antibiotic resistance. Several methods for genotypic analysis of S. pneumoniae are currently in use. The "gold standards" are pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In PFGE, the whole genome is digested with a restriction endonuclease and run slowly through an agarose gel with an alternating current (10). The fragment banding pattern produced is used to characterize the strain. PFGE has the advantage of high discrimination between strains but is relatively laborious, time-consuming, and costly and tends to be used to compare strains within a laboratory rather than between laboratories because it involves the comparison of banding patterns rather than absolute values. MLST allows a direct comparison of genotypes by comparing sequences within several housekeeping genes (4). This technique has proven extremely useful in making comparisons between strains not only within a laboratory but also internationally by using a Web-based database. Sequencing, however, is currently expensive and time-consuming.Restriction fragment length polymorphism (RFLP) analysis is often used on a PCR amplification product that is subjected to endonuclease digestion followed by comparison of the resulting banding patterns produced on an agarose gel. This technique has been performed on coding regions such as the capsule genes (1) and penicillin-binding protein genes (12). It has some of the sho...

16S rRNA Terminal Restriction Fragment Length Polymorphism for the Characterization of the Nasopharyngeal Microbiota

“…To receive the in vitro terminal fragment (TF) lengths (compared to in silico as above) of common nasopharyngeal species, chromosomal DNA was extracted from Streptococcus mitis, S. pseudopneumoniae, S. pneumoniae, S. oralis, S. gordonii, S. pyogenes, S. sanguinis, Pseudomonas aeruginosa, Rhodococcus equi, Corynebacterium pseudodiphtheriticum, Staphylococcus epidermidis, S. aureus, Haemophilus influenzae, Moraxella catarrhalis and Klebsiella pneumoniae as previously described [11], [12]. PCR reactions were performed in a total volume of 50 µl using 1×fast start Taq reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 1 µM of primer 8F FAM-AGA GTT TGA TCC TGG CTC AG and 1 µM of primer 907R CCG TCA ATT CMT TTG AGT TT, 0.2 µl of Fast start Taq Polymerase (Roche, Rotkreuz, Switzerland) and 2 µl of extracted DNA (2 ng/µl).…”

Polysaccharide Capsule Composition of Pneumococcal Serotype 19A Subtypes Is Unaltered among Subtypes and Independent of the Nutritional Environment

“…The PCR conditions were initial heat activation for 6 min at 95°C, followed by 30 cycles of 30 s at 95°C, 20 s at 60°C, and 90 s at 72°C, with a final extension of 5 min at 72°C. The reaction mixture contained 2.5 l of FastStart Taq reaction buffer without Strains were further characterized by plyNCR-restriction fragment length polymorphism (RFLP) as previously described (21). We selected the following strains for further analysis of the capsule composition ( (20,24).…”

A Universal Method for the Identification of Bacteria Based on General PCR Primers

“…A robust method, required for bacterial identification, has been perused by several investigators [4,14,26,[36][37][38][39][40]; studies on universal and multiplex primers]. With the current number of eubacterial species surpassing 7166 species [41], the UM described here [42] should fulfil this requirement.…”