ApplicationNote
Effective mechanical sample lysis for reliable pathogen
identification
Thomas Doedt, Friederike Kraemer, Marco Polidori, and Devika Mathur
QIAGEN GmbH, QIAGEN Straße 1, Hilden, Germany
In this application note, we describe the use of the TissueLyser LT, together with QIAamp® UCP Pathogen Kits, for effective
mechanical disruption of microbial samples and successful isolation of high yields of genomic DNA for pathogen
identification studies.
Introduction
Microbial DNA can be purified from various sample materials such as
whole blood swabs, cultures, and body fluids. Since a high yield of
microbial genomic DNA is crucial for reliable pathogen identification,
thorough sample lysis prior to DNA extraction is of critical importance.
Different sample pretreatment procedures can be required, depending
on the type of microbe. For some pathogens, chemical lysis may be
sufficient for sample pretreatment, while others, such as Gram-positive
bacteria or yeast and other fungi require mechanical lysis for maximal
lysis efficiency. Several different methods are available for the isolation
of genomic DNA from microbes. For successful genomic DNA extraction,
protocols not only have to be adapted to meet the requirements of different
sample types, but also to the morphology of the microorganism itself.
One of the biggest obstacles in nucleic acid isolation from bacteria and
fungi is the lysis step.

Figure 1. The TissueLyser LT ensures simultaneous disruption
of up to 12 samples.
This study describes an optimized workflow for microbial genomic DNA
isolation and highlights the importance of mechanical sample lysis for
optimal results. The benefits of using the TissueLyser LT in combination
with QIAamp UCP Pathogen Kits for sample disruption and microbial
genomic DNA purification, respectively, are demonstrated.
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QIAamp UCP Pathogen Mini Kit (without prelysis)
QIAamp UCP Pathogen Mini Kit (with prelysis)
Sample disruption was achieved using Pathogen Lysis Tubes (Pathogen
33.0
Lysis Tubes L for blood samples and Pathogen Lysis Tubes S for swabs) and
28.8
CT
Materials and methods
the TissueLyser LT (Figure 1), according to the manufacturer’s instructions.
Microbial DNA was isolated using the QIAamp UCP Pathogen Mini Kit
24.5
or the QIAamp UCP PurePathogen Blood Kit or the QIAamp DNA Mini
20.3
Kit, according to the protocols described in the respective kit handbooks.
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16.0
Swabs
Blood
Figure 2. High yield of microbial DNA through efficient cell
lysis. Cotton swabs or whole blood samples were spiked
with a mixture of cells from the indicated microbes. DNA
isolation was performed using the QIAamp UCP Pathogen
Mini Kit with or without prelysis prior to DNA isolation.
The QIAamp UCP Pathogen Mini Kit, when used in combination with Pathogen Lysis Tubes and the TissueLyser
LT, resulted in efficient cell lysis, leading to high yields of
microbial DNA, as demonstrated by the low CT values in
real-time PCR assays.
Pathogen DNA purification from swabs or 400 µl blood
Cotton swabs or whole blood samples were spiked with a mixture of
Streptococcus pyogenes, Staphylococcus haemolyticus, and Candida
albicans cells. Pathogen DNA was purified using either the QIAamp UCP
Pathogen Mini Kit alone or in combination with Pathogen Lysis Tubes
(S or L) and the TissueLyser LT. Purified DNA was quantified by performing
species-specific in-house real-time PCR assays.
Evaluating the effect of mechanical sample lysis on pathogen DNA recovery
Whole blood was spiked with purified DNA from Streptococcus pyogenes
and with viable cells from Staphylococcus haemolyticus. While the first
36
Mechanical lysis
No mechanical lysis
CT
31
half of the samples were mechanically lysed using the Pathogen Lysis
Tubes provided with the kit and the TissueLyser LT, the second half of the
samples were not treated mechanically. Microbial DNA from 8 ml of the
26
spiked blood was then purified in six replicates using the QIAamp UCP
21
PurePathogen Blood Kit, according to the protocol described in the kit
handbook. Eluates were analyzed by performing in-house real-time PCR
16
Spiked S. pyogenes
DNA (control)
S. haemolyticus
Figure 3. Efficient lysis of microbial cells and higher DNA
yield due to mechanical disruption. Whole blood was spiked
with purified DNA from Streptococcus pyogenes and with
viable cells from Staphylococcus haemolyticus. Half the
samples were mechanically lysed using Pathogen Lysis Tubes
and the TissueLyser LT and the other half of the samples were
not subjected to mechanical disruption. Microbial DNA from
the spiked blood was then purified using the QIAamp UCP
PurePathogen Blood Kit. Mechanical disruption of samples
ensured efficient cell lysis, resulting in high yields of microbial
DNA, as demonstrated by the low CT values in real-time
PCR assays.
assays specific for S. pyogenes and S. haemolyticus.
Purification of bacterial DNA from culture
DNA from Escherichia coli, Bacillus subtilis, and Streptococcus pyogenes
was purified from various volumes (0.1 ml, 0.4 ml, and 1.6 ml) of
overnight cultures using either the QIAamp DNA Mini Kit or the QIAamp
UCP Pathogen Mini Kit, alone, or in combination with Pathogen Lysis
Tubes and the TissueLyser LT. For analysis, 10 µl of the purified DNA was
run on a 1% TAE agarose gel.
www.qiagen.com
Results
The results of this study demonstrate the importance of mechanical disruption
of samples for effective lysis prior to microbial DNA purification (Figures
2–4).
Efficient sample lysis leads to high yields of DNA — crucial for reliable
pathogen identification
Pathogen identification studies require high yields of microbial DNA for
successful analysis. The yields of microbial DNA achieved are influenced
by the efficiency of sample disruption and cell lysis prior to DNA extraction.
Our results demonstrate the effectiveness of using the TissueLyser LT in
combination with QIAamp UCP Pathogen Kits for optimal microbial
DNA purification and reliable pathogen identification (Figures 2–4).
Together, these products provide a complete solution for efficient lysis of
bacteria, yeast or other fungi, and for successful purification of nucleic
acids from whole blood or other sample types — enabling reliable
pathogen identification.
QIAamp
DNA Mini Kit
QIAamp UCP Pathogen Mini Kit
No mechanical
lysis
Mechanical lysis
Pathogen
Pathogen
Lysis Tubes (S) Lysis Tubes (L)
+ TissueLyser LT
M
0.1 0.4 1.6 0.1 0.4 1.6 0.1 0.4 1.6 0.1 0.4 1.6
ml culture
E. coli
(Gram – )
B. subtilis
(Gram +)
Figure 4. Mechanical lysis ensures high yields of microbial
DNA. Bacterial DNA from the indicated volumes of bacterial
overnight cultures was purified using either the QIAamp DNA
Mini Kit or the QIAamp UCP Pathogen Mini Kit, alone, or in
combination with Pathogen Lysis Tubes and the TissueLyser LT.
High yields were achieved for samples that were subjected
to mechanical disruption. The highest yield of microbial DNA
was achieved when using the QIAamp UCP Pathogen Mini
Kit in combination with Pathogen Lysis Tubes and the
TissueLyser LT. M: marker.
S. pyogenes
(Gram +)
Sample & Assay Technologies
Conclusions
■■ High yields of microbial genomic DNA are required for reliable pathogen identification.
■■ Thorough lysis of samples prior to DNA extraction is of critical importance to ensure successful
recovery of high yields of microbial DNA.
■■ Effective sample disruption and homogenization is achieved using the TissueLyser LT.
■■ The QIAamp UCP PurePathogen Blood Kit and QIAamp UCP Pathogen Mini Kit simplify
purification of microbial DNA, delivering high yields and consistent quality.
■■ Use of QIAamp UCP Pathogen Kits in combination with the TissueLyser LT provided higher
yields of DNA (due to effective cell lysis prior to DNA purification), enabling sensitive pathogen
identification.
Ordering Information
Product
Contents
Cat. no.
TissueLyser LT*
Bead mill; requires TissueLyser LT Adapter, 12-tube
85600
TissueLyser LT Adapter, 12-Tube
Adapter for disruption of up to 12 samples in 2 ml microcentrifuge
tubes on the TissueLyser LT
69980
Pathogen Lysis Tubes S
50 Pathogen Lysis Tubes (with small beads) and 1 vial Reagent DX
19091
Pathogen Lysis Tubes L
50 Pathogen Lysis Tubes (with large beads) and 1 vial Reagent DX
19092
QIAamp UCP PurePathogen Blood Kit For 10 preps: QIAamp UCP Mini Columns, Pathogen Lysis Tubes L,
Tube Extenders (20 ml), Proteinase K, Buffers, VacConnectors,
and Collection Tubes (1.5 ml and 2 ml)
50112
QIAamp UCP Pathogen Mini Kit (50)† For 50 preps: 50 QIAamp UCP Mini Columns, Collection Tubes,
Tube Extenders, Elution Tubes, and Buffers
50214
*The TissueLyser LT must be used in combination with the TissueLyser LT Adapter, 12-Tube.
QIAamp UCP Pathogen Mini Kit does not include the Pathogen Lysis Tubes (L and S). Pathogen Lysis Tubes are sold separately.
†
The QIAamp UCP PurePathogen Blood Kit is not for use in detecting fungal nucleic acid by PCR and hybridization. For
up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual.
QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical
Services or your local distributor.
To learn more about the TissueLyser LT, visit www.qiagen.com/mechanical-lysis!
Trademarks: QIAGEN®, QIAamp® (QIAGEN Group).
1073618 08/2012
© 2012 QIAGEN, all rights reserved.
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