LOOXSTER® concentrates bacterial DNA

LOOXSTER® increases the probability of a positive 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, 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 patient’s antibiotics treatment, low abundance and non-cultivable organisms. Moreover, it takes usually 3 to 5 days to obtain a result from blood culture which is too late to initiate proper antibiotic 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 resolve the above mentioned drawbacks, the LOOXSTER® universal kit, based on SIRS-Lab’s proprietary PUREPROVE® technology, was developed. It allows the specific binding of prokaryotic DNA and removal of eukaryotic background. This results in the concentration of the prokaryotic DNA, therefore, increasing the sensitivity of the subsequent amplification methods.

What is LOOXSTER®?

LOOXSTER® universal is a kit which enables the specific concentration of prokaryotic DNA from a mixture of pro and eukaryotic DNA (see Figure 1). Due to the specific binding of prokaryotic DNA to the matrix contained in the concentration column, the unbound DNA and inhibitors are eliminated within the wash steps. The bound DNA is then eluted from the column using an appropriate buffer. The resulting isolated prokaryotic DNA is ready for use in down-stream applications such as PCR.

Fig. 1: LOOXSTER® principle. Total genomic DNA is obtained from 5 ml whole blood. The resulting nucleic acid pool contains nearly 100% eukaryotic (blue) as well as a minute quantity of prokaryotic (green) DNA. Using the PUREPROVE® technology allows the specific binding of prokaryotic DNA and the removal of more than 90% of the eukaryotic DNA. The column is then washed to remove DNA with unspecific binding and the remaining DNA is eluted. Thus, the LOOXSTER® principle concentrates the prokaryotic DNA and eliminates a high amount of background DNA, which would negatively impact on the subsequent nucleic acid-based amplification.

What’s the principle of LOOXSTER®?

Samples such as whole blood, tissue, etc. lead to a complex DNA mixture having high eukaryotic DNA background and little prokaryotic DNA. LOOXSTER® is based on the PUREPROVE® technology. This technology encompasses a protein which recognises definite motives within the prokaryotic DNA. When a complex DNA mixture is applied to a PUREPROVE® technology-based matrix, the prokaryotic DNA will bind, allowing the removal of more than 90% of the eukaryotic background DNA. The final pro- and eukaryotic DNA ratio will favour prokaryotic DNA when compared to the initial sample ratio and enables to increase the sensitivity of subsequent amplification methods.

LOOXSTER® increases the probability of a positive pathogen detection.

Removing >90% of the eukaryotic DNA contained in a sample allows to increase the statistical probability for a positive PCR and therefore, to increase the sensitivity of the resultant PCR-based detection methods. Figure 2 shows a statistical representation of the LOOXSTER® effect. The starting material, typically a clinical sample (e.g. 5 ml blood), results, on average, in 40 µg total DNA (ranging from 10-100 µg). Viewed statistically, a 40 µg sample can be divided in 100 units of 0.4 µg total DNA. A white square does not contain bacterial DNA, a grey square contains bacterial DNA. A maximum of 0.4 µg is tested in a PCR reaction. Would a PCR be performed on the starting material the probability of selecting a grey square containing prokaryotic DNA is very small.

Fig. 2: LOOXSTER® principle and statistical representation of pro- and eukaryotic DNA amounts in each fraction. Each step of the LOOXSTER® protocol results in a fraction (box with bold frame) containing different amounts of DNA as well as different ratios of prokaryotic (green) and eukaryotic DNA (blue). Each fraction can be split into 0.4 µg DNA portions containing either only eukaryotic DNA (white squares) or pro- and eukaryotic DNA (grey squares). From a statistical point of view, LOOXSTER® leads to an increased probability of a positive result.

Applying the LOOXSTER® protocol to a clinical sample will allow to bind specifically all prokaryotic DNA present in the sample (e.g. phagocytized and intracellular bacteria, free bacterial DNA) and remove the unbound DNA in the flow-through. Eluting the DNA bound on the column will therefore lead to a concentration of the prokaryotic DNA as shown in Figure 2. It can be seen that the elution increases the statistical probability for a positive PCR and therefore, the sensitivity of the test system.

PCR with & without LOOXSTER®

5 ml of whole blood spiked with group A Streptococci were lysed and total genomic DNA was extracted. The total DNA was precipitated and the pellet resuspended in 200 µl DNA-free water. 1 µl was used to determine the DNA concentration, 4 µl for real time PCR and 195 µl were applied to LOOXSTER® universal as described below.

A) LOOXSTER® universal

1. Binding step

Add 195 µl buffer D to 195 µl resuspended DNA. Quickly remove 400 µl of homogenized matrix and apply to the column. Centrifuge the column for 30 sec at 1,000 g at room temperature (RT). Discard the flow-through.

Pipette 300 µl of buffer D to the column. Centrifuge the column for 30 sec at 1,000 g at RT. Discard flow-through. Add the sample to the prepared column. Pipette the matrix-DNA-mix carefully up and down and incubate for 10 min at RT. Centrifuge for 30 sec at 1,000 g (RT).

2. Wash step

Transfer the column into a new 2 ml tube and add 2 x 300 µl of buffer D to the column. Centrifuge for 30 sec at 1,000 g (RT).

3. Elution step

Transfer the column into a new 2 ml tube and add 300 µl of buffer E. Incubate for 5 min (RT). Centrifuge for 30 sec at 1,000 g (RT). Add again 300 µl of buffer E to the column. Centrifuge for 30 sec at 1,000 g (RT). The eluate volume should be 600 µl.

4. Precipitation step

Precipitate the enriched DNA by adding 5 µl solution G, 0.1 Volume 3M NaAc pH 5.2 and 0.8 Volume Isopropanol. Short vortexing for 10 sec. Centrifuge the sample for 60 min at 16,000 g at 4°C. Discard the supernatant. Wash the pellet with 2 x 1 ml Ethanol (70%). Centrifuge 5 min at 16,000 g and discard the supernatant. Dry the pellet (RT). Dissolve the pellet in 20 µl DNA-free water.

B) Real time PCR

The samples are diluted to 50 ng / µl total genomic DNA.

SYBR Green PCR Mix (Qiagen) 5,0 µl
primer sagA fw 0,5 µl
primer sagA rev 0,5 µl
sample 4,0 µl
Sum * 10,0 µl

*200 ng total-DNA / reaction

 

Profile (on a Rotorgene3000; Corbett research):

Cycle Hold at 94°C, 15 min 0 sec.
Cycling (45 repeats)

Cycle Point
• Step 1 at 94°C, hold 30 sec
• Step 2 at 55°C, hold 30 sec
• Step 3 at 72°C, hold 60 sec, acquiring to cycling A (FAM/SYBR,ROX)

Melt (50-95°C), hold 30 sec on the 1st step,
hold 5 sec on next steps, Melt A (FAM / SYBR)

Results

The real time PCR results are presented in Figure 3 where it can be seen that using the LOOXSTER® protocol shifts the real time PCR curve away from the low copy range. The concentration obtained using LOOXSTER® is more than 10-fold.

Fig. 3: Experimental representation of the LOOXSTER® principle. 5 ml of whole blood spiked with group A Streptococci (GAS) were lysed and total genomic DNA was extracted. Real time PCR was performed on aliquots from the same blood sample before and after the LOOXSTER® sample preparation protocol. A stepwise 10-fold dilution standard curve was performed using GAS DNA from bacterial culture. Real time PCR performed on the starting material (red curve) as well as the flow-through (blue curve) led to curves lying in the low copy range between 0 and 10. However, when real time PCR was performed on DNA resulting from the LOOXSTER® sample preparation protocol, a more than 10-fold increase in sensitivity was to be noticed (green curve). Furthermore, the real time PCR curves do not lie within the low copy range anymore.

Conclusion

PUREPROVE®-based LOOXSTER® was developed to increase the sensitivity of nucleic acid-based detection methods. The use of LOOXSTER® allowed a more than 10-fold increase in signal intensity when compared to real time PCR performed on the same sample processed without the LOOXSTER® protocol. This increases the sensitivity of the method by removing the sample signal from the background noise and therefore, allows a clear assertion of the results.

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