Optimal DNA Isolation Method for Detection of Bacteria in Clinical Specimens by Broad-Range PCR

Abstract

Broad-range amplification of bacterial DNA from clinical specimens has proved useful for the diagnosis of various bacterial infections, especially during antimicrobial treatment of the patient. Optimal sample processing protocols for diagnostic broad-range bacterial PCR should release DNA from an array of target organisms with equal efficiencies and wash out inhibitory factors from various sample types without introducing bacterial DNA contamination to the amplification reaction. In the present study, two physical cell wall disintegration methods, bead beating and sonication, for enhanced detection of organisms with difficult-to-lyse cell walls were studied. The analytical sensitivities of several commercially available DNA purification kits, which were used with and without additional cell disintegration steps, were compared by using dilution series of model bacteria. Selected purification methods were used to process routine clinical specimens in parallel with the standard phenol-ether DNA extraction, and the results obtained by bacterial PCR and sequencing with the two template preparations were compared. The method with the DNA isolation kit with the lowest detection limits from the bacterial suspensions (Masterpure) did not prove to be superior to the standard method when the two methods were applied to 69 clinical specimens. For another set of 68 clinical specimens, DNA purified with a glass fiber filter column (High Pure) with an additional sonication step yielded results well in accord with those obtained by the standard method. Furthermore, bacterial DNA was detected in four samples that remained PCR negative by the standard method, and three of these contained DNA from gram-positive pathogens. Three samples were positive by the standard method only, indicating the limitations of applying any single method to all samples.Direct amplification of bacterial DNA from clinical specimens with broad-range primers provides an alternative approach to the recognition of pathogens infecting normally sterile body compartments. In comparing the molecular diagnosis obtained by broad-range bacterial PCR and partial sequencing of the amplicon to the results of routine bacterial cultures, we found 83% overall agreement for a set of 536 clinical specimens from hospitalized patients (7). The molecular approach proved superior to culture during antimicrobial treatment of the patient and in detection of bacteria with unusual growth requirements. A major drawback of the molecular method in comparison to bacterial culture was the difficulty in detecting species with gram-positive cell walls and mycobacteria. This was associated with problems in breaking bacterial cell walls and releasing bacterial DNA for amplification when standard phenol-ether DNA extraction was used. This finding led us to search for better methods to prepare the clinical specimens for broad-range bacterial PCR assay.In general, an optimal sample processing method should concentrate the DNA, especially that derived from the target organism, and wash out in...

Evaluation of microbial RNA extractions from Streptococcus pneumoniae

“…The coupled efficiency of two methods for RNA quantification, PCR and spectrophotometry, was also determined. Physical cell wall disintegration by sonication was previously described for RNA isolation in organisms with difficult-to-lyse cell walls (Frostegard et al, 1999;Rantakokko-Jalava et al, 2002), but in the present study, this method was not effective for isolating SP RNA. Enzymatic digestion by mutanolysin treatment or mutanolysin combined with proteinase K and SDS was also reported to yield high quality RNA and DNA from gram positive bacteria (Ampe et al, 1998;Fliss et al, 1991;Gopalakrishna et al, 1981;Shiroza et al, 1998;Simpson et al, 1993;Zhou et al, 1996), but again, the present study documented only a low RNA yield for these procedures and suggested that the required sample incubations at 50 o C ~ 37 o C may have been responsible by causing significant RNA degradation.…”

An Efficient DNA Extraction Method for Lactobacillus casei, a Difficult-to-Lyse Bacterium

“…P2 and P3 protocols had high-purity products (A260/A280 1.81 and 1.92, respectively). This may be due to the use of high concentration of lysozyme (50 mg/mL) for P2 protocol and the additional step of protein precipitation and RNase (20 mg/mL) for P3 protocol, which may have resulted in the removal of contaminants and increased the purity, similar to the previously described investigations (10,21). The purity of DNA in all kit-based protocols was ~1.50 which was lower than that of the traditional protocols.…”

Methodological variations in the isolation of genomic DNA from Streptococcus bacteria

“…2). This work demonstrated that molecular detection of bacteria with resistant cell walls in clinical samples could be enhanced by the use of physical cell wall desintegration methods as related by others authors (Rantakokko-Jalava and Jalava, 2002). Results also indicated that no single method was optimal for the detection of all bacteria that might be present in biological fluids, and the use of more than one method should be recommended.…”

“…The cell wall of Gram-positive bacteria can be broken by the use of enzymes that degrade peptidoglycans such as the lysozyme and mutanolysin present in the commercial kits (Yano et al, 2002;Old et al, 2006), which cause the method to be expensive. The use of cheaper alternative methods is not always appropriated given the difficulty in breaking the cell wall when some reagents are used, such as phenol-ether (Rantakokko-Jalava and Jalava, 2002). In this sense, bacterial DNA can be extracted from sera by boiling without using any toxic reagent, overcoming the use of expensive commercial kits for DNA purification (Mayoral et al, 2005).…”