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RTP® Bacteria DNA Mini Kit

Overview
Applications
Manual
MSDS
References

The RTP Bacteria DNA Mini Kit contains the pre-filled Extraction Tubes with lyophilized lysis components for genomic DNA isolation from different bacteria species from a broad range of starting materials. After lysis, the samples are transferred to a spin column-based procedure with a PCR template preparation time of 25 minutes.

Starting Material

bacteria pellets, tissue samples, biopsy material, paraffin embedded tissue, blood, cell-free body fluids, urine, paper points, swabs, water

Product Characteristics

Product	Starting Material	Average Yield	Preparation Time
RTP® Bacteria DNA Mini Kit	max.10⁹bacteria, 1-10 mg tissue, 1-100 µl whole blood, 15-50 ml urine, up to 1 l water	depending on the starting material	25 min (without lysis time)

Advantages

time saving and safer sample handling: 60 % less pipetting steps and tip consumption
one lysis tube: Ready To Prep (RTP): pre-filled Extraction Tube with lyophilized lysis components (lysis buffer, Lysozyme, Proteinase K), carrier RNA, and internal DNA extraction control; stable at room temperature
environment-friendly - less infectious plastic waste due to reduced hands-on steps
for a wide variety of samples
CE-marked in compliance with the Directive 98/79/EC on in vitro diagnostic medical devices (IVD-Directive)*

Overview

Using the RTP Bacteria DNA Mini Kit all types of samples are transferred to the Extraction Tube together with a specially designed Resuspension Buffer. After cell wall lysis of bacterial cells using Lysozym, provided in the Extraction Tube, the pre-filled buffer and Proteinase K lyse the material and digest the proteins and stabilize the nucleic acid by inactivating intracellular DNases. This is followed by the addition of a binding buffer to enhance selective DNA adsorption to the membranes. The membranes are efficiently washed before the DNA is eluted to remove all contaminants. High extraction efficiency and detection sensitivities are realized. All kit components can be stored at room temperature. The purified high quality DNA is ready to use for subsequent downstream applications or can be stored at –20 °C for subsequent use:

PCR
real-time PCR (quantitative PCR, like TaqMan and LightCycler technologies)
RFLP analysis

*) Products which are CE-marked according to the IVD-Directive can be used for diagnostic applications in countries where this directive is recognized.

Ordering Information

Product	Package Size	Catalogue No
RTP Bacteria DNA Mini Kit	50 purifications	1033200200 (old: 10332002)
RTP Bacteria DNA Mini Kit	250 purifications	1033200300 (old: 10332003)

Process Overview

Applications

Identification of MRSA isolates

Identification of MRSA isolates by multiplex-PCR amplification of three target gene sequences (23S rDNA, nuc, mec). Bacterial genomic DNA was isolated using RTP Bacteria DNA Mini Kit according to the standard protocol. Amplificates were analyzed on a 2.0 % agarose gel.

Data kindly provided by A. Fetsch, Federal Institute for Risk Assessment (BfR), National Reference Laboratory for coagulase positive staphylococci including Staphylococcus aureus, Berlin, Germany

Specific detection of periodontal pathogens from paper points using TaqMan analysis

Four teeth of a patient were sampled with one paper point, respectively. The bacterial DNA was isolated using RTP Bacteria DNA Mini Kit. All four samples were positive, although sample 3 harbored hardly bacterial DNA.

A) PCR products for the analysis of polymicrobial infections using DHPLC (Domann et al., 2003).
Sample 1 = tooth 27 mb
Sample 2 = tooth 37 mb
Sample3 = tooth 45 db
Sample4 = tooth 46 mb.
Size of amplicons: ~470 bp. MW=Marker 9 (Roche) - mb=mesiobuccal, db=distobuccal.
B) Specific TaqMan PCR to detect Prevotella intermedia (Kuboniwa et al., 2004). Samples 1, 2, and 4 were positive.
C) Specific TaqMan PCR to detect Treponema denticola (Kuboniwa et al., 2004).

Data kindly provided by Dr. E. Domann, Institute of Medical Microbiology, University of Giessen, Sampling: Medical Centre of Odontology, University of Giessen, Germany.

Detection of periodontal pathogens from paper points using DNA Microarray analysis

Detection of periodontal pathogens with the ParoCheck 20 DNA Microarray (Lambda GmbH and Greiner Bio-One GmbH). The bacterial DNA from paper points from teeth were purified with the RTP Bacteria DNA Mini Kit.

quality control

Printing Control: 3 of 3 spots have SNR larger than 50
Positive Control: 6 of 6 spots have a SNR larger than 2
Sensitivity: Dilution Factor SNR
Hybridization Control: 6 of 6 spots have a SNR larger than 2
Sensitivity: Dilution Factor SNR

bacterial results

Actinobacillus actinomycetemcomitans, A.a. negative Fusobacterium nucleatum, F.n. positive
Actinomyces odonotolyticus, A.o. positive Peptostreptococcus micros, P.m. positive
Actinomyces viscosus, A.v. positive Porphyromonas gingivalis, P.g. positive
Bacteriodes forsythus, B.f. positive Prevotella intermedia, P.i. positive
Campylobacter, C.c. positive Prevotella nigrescens, P.n. positive
Campylobacter gracilis (Bacteriodes g.), C.g. positive Streptococcus constellatus, S.c. positive
Campylobacter rectus, C.r. positive Streptococcus gordonii, S.g. positive
Capnocytophaga ginigivallis, Cap.g. negative Streptococcus mitis, S.m. positive
Elkenella corrodens, E.c. negative Treponema denticola, T.d. positive
Eubacterium nodatum, E.n. positive Veilionella parvula, V.p. positive