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Looxster® - Enrichment and isolation of bacterial DNA using Pureprove® Technology
Sample preparation for pathogen detection

SIRS-Lab GmbH, Winzerlaer Str. 2, D-07745 Jena

Introduction

Detection of the bacterial content of a specific sample, irrelevant from its source (i.e. Clinical, Food or Environmental), is not as straightforward as it seems. In clinical microbiology laboratories, the gold standard method for the detection of pathogens in patients suspected of systemic infections is the blood culture. However, this technique is known to have many drawbacks especially with regard to non-cultivable organisms, low abundance (longer lag phase) and patient’s antibiotics treatment. Moreover, it takes usually 3 to 5 days to obtain a result from blood cultures which is often too late to initiate proper antibiotic-based therapy. Amplification-based methods such as PCR allow results in a more rapid fashion. However, their sensitivity to date are no better, and sometimes worse, than culture-based methods. To increase the sensitivity of nucleic acid based detection methods SIRS-Lab developed LOOXSTER _^® universal. LOOXSTER^® universal based on SIRS-Lab’s proprietary PUREPROVE^® technology allows the specific binding of prokaryotic DNA. LOOXSTER^® universal was designed for the specific culture- independent enrichment and isolation of bacterial genomic DNA from various origins and is compatible with any of the current nucleic acid amplification and/or hybridisation methodologies. LOOXSTER^® universal’s performances were tested using whole blood from ICU patients and the bacterial genomic DNA signals obtained by PCR for samples prepared with and without the use of LOOXSTER^® universal were compared. These results were then compared with those obtained from blood culture experiments.

Experimental procedures (see figure 1)

Sample collection: After approval of the study protocol by the local Ethical Committee, blood samples were obtained from 21 patients (8 females, 13 males, median age 65 yrs, range 34-82 yrs) who were admitted to an operative ICU and were suspected to suffer from an infection based on clinical assessment (e.g. prescription for blood cultures).

Performing this protocol from the sample preparation to the detection step requires less than 8 hrs and fits easily in a day’s work.

Fig. 1: Schematic view of the study’s protocol.

Total genomic DNA extraction

Total genomic DNA extraction (i.e. human and bacterial DNA) was performed from each patient’s Buffy coat. At first, leukocytes and/or bacteria cells were pelleted by centrifugation and resuspended in 200 µl TE- buffer. Further, a mixture of cell wall lytic enzymes was added to break open the bacteria. After an incubation at 37°C for 90 minutes, the remaining cells were finally lysed using 50 µl of 10% SDS and 50 µl proteinase K (20mg•ml-1) for 2 hours at 50°C. Total genomic DNA was extracted using the phenol-chloroform-isoamylalcohol (25:24:1) method. Furthermore, DNA was precipitated using 700 µl of ice-cold isopropanol. Precipitated genomic DNA were then recovered by centrifugation (15,000 x g for 30 minutes), washed twice in 70% ice-cold ethanol (5 minutes, 15,000 x g) and vacuum-dried.

Bacterial genomic DNA enrichment

Separation of bacterial DNA from an excess of human DNA

Specific bacterial genomic DNA was obtained using SIRS-Lab’s LOOXSTER^® universal. LOOXSTER^® universal contains columns, enrichment matrix, collection tubes and reagents to perform the enrichment of prokaryotic DNA from human/bacterial DNA mixed samples. The protocol can be summarised as follows. 50 µl of total genomic DNA (i.e. human and prokaryotic DNA) were applied to an enrichment column, vortexed and incubated for 1 min at room temperature. The column was then centrifuged at 15,000 x g for 30 sec and the flow-through (F) stored on ice until use. 100 µl of wash buffer were pipetted on the column, followed by vortexing, centrifuged at 15,000 x g for 30 sec and the flow-through (W1) stored on ice until use. The bacterial DNA was then eluted from the enrichment column using 100 µl Elution buffer 1, followed by vortexing, centrifugation (E1) and stored on ice until use. This step was repeated using Elution buffer 2, giving (E2).

Bacterial DNA precipitation

The fractions F, W1, E1, E2 each received 0.7 volume (i.e. 700 µl) isopropanol. The DNA was pelletted by centrifugation at 15,000 x g for 30 min. The supernatants were discarded and the pellets washed twice in 70% ethanol, vacuum dried, resuspended in 30-50 µl TE and stored at -20°C until use.

Amplification by PCR

Primers - Universal 16S primers were designed in order to obtain an annealing temperature of 53°C and an amplicon length of 994 bp as followed:

PCR amplification - PCR was performed using the gen amp PCRSystem 2400 from Perkin Elmer (Fremont, USA) in 50 µl reaction volumes consisting of 5 µl total genomic DNA, 10 pmol of each forward and reverse primer, 0.2 mM from each dNTP, 5 µl of 10x Mix (MBI Fermentas, St. Leon- Rot, Germany), 2.5 mM MgCl2, (MBI Fermentas, St. Leon- Rot, Germany) and 1U/reaction Taq polymerase (MBI Fermentas, St. Leon- Rot, Germany). An initial denaturation of the DNA was carried out at 96°C for 10 min followed by 40 cycles of 96°C for 30 sec, 53°C for 30 sec, 72°C for 3 min. Each sample as well as a negative template control (NTC), a positive control (Staphylococcus aureus DNA as template) and an inhibition control (total genomic DNA + Staphylococcus aureus DNA) were performed.

PCR analysis - After completion of the PCR, amplicons were resolved on a 2% agarose gel stained with SYBR - Green and visualised under UV and documented by gel documentation system.

Results

The impact of the sample preparation using LOOXSTER^® universal was compared to results obtained from PCR with and without the up-stream use of the LOOXSTER^® universal as well as blood culture (data provided by the department of Clinical Microbiology, FSU, Jena, Prof. Dr. Straube) (Figure 2).

Fig. 2: Comparison of results after 16S rRNA gene PCR between purified DNA samples using LOOXSTER® universal and DNA isolated from patient’s Buffy coat. In panel A: F: Flow-through Fraction, W: Wash-Fraction, E1: Elution buffer 1, E2: Elution buffer 2. In panel B: DNA: DNA isolated from Buffy coat (without the use of LOOXSTER® universal), I: Inhibition control (without the use of LOOXSTER® universal). In panel C: P: Positive control, N: Negative control, S: Standard.

In figure 2, three distinctive groups of results demonstrate the functioning of LOOXSTER^® universal:

Panel A shows the specific binding, elution and detection of bacterial genomic DNA in the elution fractions and not in the flow-through or wash fraction.

Panel B validates the proper functioning of the PCR protocol and the absence of amplification inhibitors. Moreover, when the same PCR protocol was performed on DNA that were not processed using LOOXSTER^® universal, no signal could be detected.

In Panel C, as expected, the negative control did not show any contamination. The positive demonstrates the proper functioning of the PCR protocol and amplification of the desired amplicon. It is therefore to note that prokaryotic DNA can be successfully released upon addition of the elution buffers as well as amplified. Moreover, when using the same amount of starting material, the specific 16S amplicon can only be detected when using LOOXSTER^® universal, demonstrating the efficient isolation, enrichment and purity of prokaryotic DNA as well as an increased sensitivity due to the use of LOOXSTER^® universal when compared PCR with and without LOOXSTER^® universal.

Fig. 3: Pathogen presence / absence determined by blood culture, PCR w/o LOOXSTER® universal and PCR with LOOXSTER® universal.

Figure 3 shows that performing PCR directly on the DNA obtained from the different patients’ blood samples did not significantly enhance sensitivity when compared to blood culture, as the number of positives and negatives are similar. The minute quantity of prokaryotic DNA present is drawn in an excess eukaryotic DNA, therefore making the prokaryotic DNA concentration fall under the limit of detection of the PCR technique. However, when comparing the PCR results obtained with and without LOOXSTER^® universal, it can be seen that the sensitivity is greatly improved. This correlates with clinical data that suggested infection for all these patients. LOOXSTER^® universal enabled not only the separation of prokaryotic DNA from an excess eukaryotic DNA but also its concentration and successful amplification by a classic PCR methodology.

Conclusions

These experiments aimed at demonstrating a distinct improvement in the detection of bacterial genomic DNA from complex samples such as blood as well as establishing a more rapid and sensitive detection method when compared to blood culture.

This study demonstrated the impact of LOOXSTER^® universal on the amplification protocol as well as when compared to standard culture-dependent methods. LOOXSTER^® universal leads to a drastic increase in sensitivity, as well as savings in time and reagents.

Acknowledgments

Experiments were performed and data provided by the Institute for Medical Microbiology, Friedrich-Schiller-University Jena, Semmelweisstrasse 4, Jena, Germany.