Research Products and CE IVD Products

Brochure 2009/2010

Bacterial and Fungal DNA Isolation from Blood DNA-Free PCR Reagents PCR Assays for Pathogen Detection

Order: +49(0)-421-22 09 77 70

Molzym GmbH & Co. KG

Molzyms R&D activities are directed towards producing innovative solutions for the molecular detection and identification of pathogens. Our developmental research is particularly focussed on new enzymes and processes that facilitate and enhance the molecular diagnosis of diseases caused by microorganisms. Among Molzyms latest products are pre-analytical kits, diagnostic reagents and assays for the screening and monitoring of bacterial as well as fungal agents of septicemia. The pre-analytical kits, MolYsis, are for the enrichment of bacterial DNA from whole blood, blood culture and other body fluids. Molzyms DNA-free reagents for PCR analysis add to a package of products for assaying pathogens in blood. A variety of products offers the opportunity to set up individual approaches to accurate results, without false signalling, in bacterial pathogen detection. The product portfolio ranges from CE-marked DNA-free Taq DNA polymerase for in vitro diagnostic use, MolTaq 16S, to various DNAfree mastermix designs, Mastermix 16S product series. A further product for in vitro diagnostic use in the routine lab is SepsiTest combining innovative technologies for bacterial and yeast DNA isolation and universal rDNA detection in one kit. If specific solutions are requested, Molzym offers a service for the purification of customised mastermixes. Molzym is always open to any collaboration directed to the development of individualised pre-analytical and analytical adaptations, including automated DNA isolation.

For detailed information, application guides and additional services, please visit www.molzym.com and www.sepsitest.com or contact us at support@molzym.com

order@molzym.com

Molzyms sepsis PCR diagnostic products...............................................................................6

icity

Chapter 3: MolTaq 16S and Mastermix 16S DNA-free Reagents for the PCR detection of Bacteria...........................................................23

rmix Chapter 4: SepsiTestTM- CE IVD Pathogen Detection Kit................................................................3

SepsiTestTM - complete system of targeted bacterial and yeast DNA PCR...

-69 61 Order: +49(0)-421-22 09 62 77 0 70

Chapter 1: Introduction to Molzyms Molecular Sepsis Diagnostics Products

order@molzym.com

Chapter 1:

1
Chapter 1: Introduction to Molzyms Molecular Sepsis Diagnostics Products 

Molzyms sepsis PCR diagnostic products...........................................................6

Order: +49(0)-421-69 61 62 0

1
Need for rapid sepsis diagnosis

Chapter 1: Introduction to Molzyms Molecular Sepsis Diagnostics Products 

Sepsis is a serious medical condition characterised by a whole-body inflammatory state caused by infection. Symptoms of sepsis are often related to the underlying infectious process. When the infection crosses into sepsis, the resulting symptoms are that of systemic inflammatory response syndrome (SIRS), characterised by general inflammation, fever or hypothermia, abnormal white blood cell count, and raised heart rate (tachycardia) and breathing rate (tachy pnea). The immunological response that causes sepsis is a systemic inflammatory response causing widespread activation of inflammation and coagulation pathways. This may progress to dysfunction of the circulatory system and, even under optimal treatment, may result in the multiple organ dysfunction syndrome and eventually death. Immediate source control, particularly early antibiotic therapy, remains the cornerstone of care in severe bacterial infection. Recent data shows that lethality almost doubles if an antibiotic therapy is not tailored to the specific pathogen or its resistance pattern. The mortality in creases by approximately 5% per hour when the start of the therapy is delayed. Blood culture, todays golden standard identifying causative organisms, is hampered by low sensitivity and even when positive, the results are obtained too late to influence clinical decision making in the early hours after onset of sepsis. More than 751,000 cases of severe sepsis occur in the United States of America each year (Angus et al. 2001). Septic diseases are a major cause of death in intensive care units, with a mortality rate of 215,000 per year. With a mortality ranging between 28-50%, sepsis is a major cause of death worldwide, killing approximately 1,400 persons every day. Sepsis is a clinical syndrome characterised by the presence of both an infection and a systemic inflammatory response. The decisive factor for the development of an organ dysfunction and, thus, for the patients prognosis is the deregulation of the endogenic defence and repair systems. There is, therefore, an urgent need to reduce the uncertainty of todays diagnosis and, in so doing, to reduce the mortality rate of septic patients. Molecular biological techniques, in particular PCR-based methods, are generally accepted as a promising means of early sepsis diagnosis. Molzym has developed a portfolio of products adapted to the needs of sepsis research and routine diagnosis. In the following chapters Molzyms products specialised for the detecting and monitoring of pathogens in whole blood and blood culture are described. The products comprise the whole pathway of nucleic acid-based solutions, ranging from pre-analytics to PCR assaying of pathogens.

order@molzym.com

1
Molzyms sepsis PCR diagnostics products for research use
The golden standardof diagnosis of sepsis is blood culturing. The detection of pathogens by blood culture is time-consuming, generally taking 1 day, for fastidious organisms even several days. Further, non-growing pathogens are not detected by blood culturing. Molecular biology methods, in particular PCR, provide promising additional diagnostic tools for sepsis diagnosis, mainly because results are obtained within only 4 to 8 hours. Septic diseases are caused by a variety of microorganisms, mainly Gram-positive and Gramnegative bacteria. PCR diagnosis, therefore, demands assays detecting the broad range of pathogens involved. With its portfolio of research and development products and complete systems for routine diagnostics, Molzym provides solutions for the rapid, reliable and sensitive detection of pathogenic microorganisms in the blood stream. Molzym offers single components from pre-analytics to PCR, customised solutions for the development of assays as well as universal 16S rDNA detection solutions. Pathogen identification by sequencing of the amplification product and online searching of gene data libraries (www.sepsitest-blast.net) can round up diagnosis of sepsis and provide information for rapid therapy. Total processing of blood samples from targeted pathogen DNA isolation to pathogen detection by Molzyms SepsiTest takes only approx. 4 hours, identification by sequence analysis included only about 6 hours. All Molzym products are manufactured under CE-conforming conditions which exclude the contamination of reagents with exogenous bacterial DNA. Molzyms technology guarantees the removal of bacterial DNA from enzymes, PCR reagents, including Taq DNA polymerase, and buffers of pre-analytic and diagnostic kits. All Molzym reagents and kits, except CE IVD SepsiTest, are for general laboratory use only and NOT for in vitro diagnostic procedures. Overview of Molzyms products for PCR detection of sepsis-causing pathogens in blood

Reference: D.C. Angus, W.T. Linde-Zwirble, J. Lidicker, G. Clermont, J. Carcillo, M.R. Pinsky (2001). Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit. Care Med. 29: 1303-1310

Order: +49(0)-421-69 61 62 0

Chapter 1: Introduction to Molzyms Molecular Sepsis Diagnostics Products 

Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation

order@molzym.com

Order: +49(0)-421-69 61 62 0

Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation

2
Introduction to the Technology Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 
Human DNA causes loss of PCR detection sensitivity and specificity
The performance and detection limits of PCR assays for the diagnosis of septic disease are strongly dependant on the method of DNA preparation. The extraction of total DNA from blood generates a mixture of human and bacterial DNA. A problem with this approach is that human DNA can provide non-specific binding sites for bacteria-specific primers. In particular, with high human to bacterial DNA ratios, non-specific primer binding can lead to false-negative and false-positive results and hence to a loss of sensitivity and specificity of the assay (Navarro et al. 2002). Apparent loss of specificity can be observed in a universal 16S rDNA PCR reaction as the result of the co-amplification of human sequences (Fig. 1b). In the example, a 3,230-fold mass excess of human to bacterial DNA led to complete failure in the sequence identification analysis. Furthermore, in the absence of bacterial DNA, a false positive signal was produced by the amplification of a human gene (Fig. 1c).

Fig. 1: Influence of human DNA on the 16S rDNA detection of bacterial targets in a PCR reaction (Disqu, 2007). PCRs were run using Mastermix 16S Basic with primers RWO1-DG74 (Greisen et al. 1994) and analysed using gel electrophoresis. PCR products were sequenced, and a similarity search was performed using the BLAST analysis tool. a) 13 pg of P. aeruginosa (P.a.) DNA as target; b) mixture of P. aeruginosa DNA (13 pg) and human DNA (hum.) (42 ng, giving a 3,230 fold mass excess); c) 42 ng human DNA. The amount of human DNA applied in the experiment corresponds to the amount usually extracted from 0.2 ml blood. The amount of bacterial DNA equals approx. 2000 cells.

order@molzym.com

2
Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 10

Non-specific binding of primers to human DNA decreases the concentration of free primers available for binding to the bacterial target. An experiment, in which human and bacterial DNA was included in a universal 16S rDNA Real-Time PCR (qPCR) assay (Mastermix Complete), demonstrates the effect of non-specific primer binding on the detection of bacteria (Fig. 2).

Fig. 2: Loss of sensitivity in the 16S rDNA detection of bacterial targets by primer binding to human DNA (Disqu, 2007). Real-Time PCR runs were performed using Mastermix 16S Complete. Left graph: documentation of the amplification; arrows indicate the shift in C(T) values as a result of human DNA (hDNA) in the assay containing P. aeruginosa DNA (P. a.) as a target. Right graph: plot of C(T) values vs. the amount of bacterial DNA in the absence and presence of a constant amount of hDNA (42 ng).

In the presence of human DNA a shift in the crossing point to approximately 3 C(T) values higher than with only the bacterial target occurred (Fig. 2, left graph). This corresponds to a detection sensitivity approximately one order of magnitude lower. The negative effect of human DNA on the sensitivity of bacterial target detection was observed over a range of more than two orders of magnitude in the amount of bacterial DNA (Fig. 2, right graph).

Order: +49(0)-421-69 61 62 0

2
MolYsis - A tool for human DNA removal

Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 11

MolYsis provides a tool which, in an easy and highly efficient way, removes human DNA and enriches bacterial DNA from blood. MolYsis thereby enhances the diagnostic sensitivity and specificity of pathogen detection with regard to false signals from human DNA. A special feature of MolYsis is that pediatric (0.2 ml) as well as medium (1 ml) and large blood volume samples (5 ml) are processed in a convenient and rapid Mini DNA isolation format. Also, automated systems can be connected downstream of the MolYsis Basic treatment.

Fig. 3: The MolYsis procedure for human DNA removal and bacteria enrichment from whole blood. Left image: Sketch of the procedure. Right picture: Gel electrophoretic analysis of bacteria-spiked blood extracts demonstrating the degradation of human DNA (MolYsis Basic + Qiagen QiaAmp DNA Blood Mini Kit). Reference: total DNA extracts (QiaAmp); US: unspiked sample, WBC: white blood cell

order@molzym.com

2
Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 12
In only 3 steps the MolYsis procedure effects the selective lysis of blood cells (Fig. 3, step 1) followed by the quantitative degradation (>99 %) by a proprietary DNase, MolDNase, of released human DNA (step 2). Finally (step 3), the bacteria are enriched from the lysate by centrifugation, resuspended in a small volume of buffer and treated with BugLysis, a reagent optimised for the lysis of both Gram-negative and Gram-positive bacteria, including staphylococci, streptococci and enterococci (Tab. 2). After this point commercial Mini isolation procedures for bacterial DNA are used.

The effect of MolYsis on the detection of bacteria in blood using PCR

The effect of MolYsis treatment of blood on the detection of bacteria is an increase in diagnostic sensitivity and specificity. An example of the sensitivity increase produced by MolYsis treatment is shown in Table 1. In a universal 16S rDNA PCR assay (Mastermix 16S Complete), detection of S. aureus is possible at considerably lower bacterial loads when blood is subjected to MolYsis treatment before DNA extraction (Table 1, B-D) than when total DNA is isolated (Table 1, A). MolYsis treatment applied to a small blood volume, such as a pediatric sample, with Qiagen QiaAmp DNA isolation gave a 1,000-fold increase in detection sensitivity (Table 1, B). In addition, PCR detection sensitivity was further increased by 5,000fold when 5 ml blood was treated by the MolYsis Basic5 kit and bacterial DNA isolated using QiaAmp (Table 1, C). Using MolYsis Complete5, the kit for human DNA removal and bacterial DNA isolation combined, resulted in a 20,000-fold increase in detection sensitivity.
Table 1: Increase in sensitivity of PCR detection by MolYsis treatment of whole blood (spike experiments) (Disqu, 2007)

Kit

extracted DNA human + S. aureus

blood volume (ml) 0.2

PCR detection limit (CFU/ml) ** 1.6 x 106

sensitivity factor

A) QiaAmp* Blood DNA Mini B) MolYsis Basic + QiaAmp* Blood DNA Mini C) MolYsis Basic5 + QiaAmp* Blood DNA Mini D) MolYsis Complete5
* **

S. aureus

0.2

1,600

1,000

S. aureus S. aureus

5.0 5.0

320 80

5,000 20,000

QiaAmp is a trade name of Qiagen Mastermix 16S Complete assay

Order: +49(0)-421-69 61 62 0

2
Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 13
Besides the reagents for human DNA removal BugLysis is a component of all MolYsis kits; it is crucial for maximising the yields of pure bacterial DNA. BugLysis is an optimised reagent that effectively degrades cell walls of Gram-negative and Gram-positive bacterial pathogens (Table 2). BugLysis is used in combination with proteinase K and lysis buffer, causing the lysis of bacterial cells, including encapsulated pathogens. After purification using Mini spin column kits the highest yields of pure bacterial DNA are obtained from blood for sensitive PCR detection assays.

Table 2: Bacterial strains evaluated for cell wall hydrolysis by BugLysis *

Gram-positive Bacillus subtilis, Bacillus cereus, Corynebacterium diphteriae, Enterococcus faecalis, Entero coccus faecium, Lactobacillus sp., Micrococcus luteus, Mycobacterium phlei, Staphylococcus aureus, Staphylococcus carnosus, Staphylococcus epidermidis, Streptococcus agalactiae (Sero-Group B), Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes (Sero-Group A), streptococci (Sero-Group G) Gram-negative Acinetobacter sp., Brevundimonas sp., Escherichia coli, Enterobacter aerogenes, Serratia marcescens, Klebsiella pneumoniae, Massilia sp., Neisseria meningitis, Neisseria subflava, Porphyromonas gingivalis, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas stutzeri

* 108-109 cultured cells were treated with and without BugLysis (control) and DNA was isolated using Molzyms MolYsis Complete5 kit. DNA amounts were estimated by comparison to DNA standards in an agarose gel electrophoretic analysis.

MolYsis was tested using blood samples of septicemic patients. DNA extracts, prepared by Qiagen QiaAmp kit and MolYsis Basic together with QiaAmp, respectively, were analysed by PCR amplification of the 16S-23S rDNA internal transcribed spacer region. With total DNA extracts (QiaAmp) a variety of bands were observed as the result of human sequence amplification (Fig. 4, lanes C and D). After Molysis treatment and DNA extraction using QiaAmp, all of the human-specific PCR signals vanished as the result of human DNA degradation. New bands appeared indicating the presence of bacterial cells in the blood samples (comparison to standard band patterns suggested S. aureus, not shown). In summary, the removal of human DNA by MolYsis treatment combined with cell wall hydrolysis of enriched bacterial cells by the BugLysis reagent ensures the isolation of pure bacterial DNA and thus a highly sensitive PCR detection of pathogens in blood samples.

order@molzym.com

2
Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 14
Fig. 4: DNA extracts from 0.2 ml blood samples of two septicemic patients (A, C resp. B, D) (Handschur et al. 2008). 16S-23S rDNA internal transcribed spacer PCR analysis; long arrows: human signals with Qiagen QiaAmp total DNA extracts; short arrows: bacterial signals using MolYsis Basic and Qiagen QiaAmp DNA extracts; N: negative PCR control; M: DNA size standard.

Bacterial DNA isolation from blood culture

A goal of sepsis patient treatment is directed therapy at the earliest point possible. Detection of pathogens by blood culture analysis is time-consuming, taking at least 24 hours. An approach which employs molecular methods, including PCR, combined with blood culture analysis will provide differentiation results within a few hours instead of another day or more by microbio logical methods. Also, slowly growing strains can be detected and identified by PCR before positive signals arise in the blood culture (Gebert et al. 2008).. A problem with the extraction of DNA from blood cultures is the co-elution of PCR inhibitors such as polyanetholesulfonate (SPS) (Fredricks and Relman 1998). Another problem comes from the presence of bacterial DNA in uninoculated blood culture media (Fredricks and Relman 1998, Millar et al. 2000) and human DNA from blood cells interfering with pathogen analysis by general 16S rDNA PCR. MolYsis Plus provides a means of efficient removal of PCR inhibitors and human DNA and thus allows the reliable detection of low bacterial titres before a positive blood culture reaction (Gebert et al. 2008). In a case study, a total of 72 blood cultures was analysed using DNA prepared with MolYsis Plus and (i) 16S rDNA PCR (Mastermix 16S Basic and universal 16S rDNA primers (Weidner et al. 1996) and sequencing (700 b) and (ii) 16/23S intergenic sequence PCR polymorphism analysis (Mendoza et al. 1998). The data were compared with microbiological identification results.

Order: +49(0)-421-69 61 62 0

2
Table 3: Case study of blood cultures using MolYsis Plus/16S rDNA PCR/sequencing

Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation

Result / species Negative Positive, total incl. 2 strain cultures Single strain cultures: S. aureus E. faecalis S. epidermidis E. coli P. mirabilis K. pneumoniae Others

MolYsis Plus and PCR Microbiology 24 24 48 48 4 3

12 9 8 7 3 2 3

12 9 7 6 3 2 6

The results (Tab. 3) indicate a concordant number of negative and positive results obtained with MolYsis Plus/PCR/sequencing and microbiological analysis. Sequencing of PCR products gave a clear indication of the removal of contaminating DNA from the culture and the human cells that can interfere with strain identification. The 4 most prominent strains with similar frequencies in both analyses were S. aureus, E. faecalis, S. epidermidis and E. coli. A similar number of blood cultures containing 2 strains was observed with both diagnostic methods. MolYsis Plus eliminates PCR inhibitors and human DNA very efficiently from blood culture and generates pure bacterial DNA. The detection of pathogens by PCR using the MolYsis Plus sample preparation kit gives the opportunity to generate reliable and quick data sets.

15

order@molzym.com

2
References:

S. Gebert, D. Siegel, N. Wellinghausen (2008) Rapid detection of pathogens in blood culture bottles by real-time PCR in conjunction with the pre-analytic tool MolYsis. J. Infect. (2008) 57: 307-316. C. Disqu (2007). Einfluss der DNA-Extraktion auf die PCR-Detektion von Sepsiserregern. BIOspektrum 06: 627-629. K. Greisen, M. Loeffelholz, A. Purohit and D. Leong (1994). PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J. Clin. Microbiol. 32: 335-351. M. Handschur, H. Karlic, C. Hertel, M. Pfeilstcker, A.G. Haslberger (2009) Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood. Comp. Immunol. Microbiol. Infect. Dis. 32: 207-219. S. Gebert, D. Siegel, N. Wellinghausen (2008) Rapid detection of pathogens in blood culture bottles by real-time PCR in conjunction with the pre-analytic tool MolYsis. J. Infect. (2008) 57: 307-316. D.N. Fredricks, D.A. Relman (1998). Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J. Clin. Microbiol. 36: 2810-2816. B.C. Millar, X. Jiru, J.E. Moore, J.A.P. Earle (2000). A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material. J. Microbial Meth. 42: 139-147. S. Weidner, W. Arnold, A. Phler (1996). Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes. Appl. Environ. Microbiol. 62: 766-771. M. Mendoza, H. Meugnier, M. Bes, J. Etienne, J. Freney (1998). Identification of Staphylo coccus species by 16S-23S rDNA intergenic spacer PCR analysis. Int. J. Syst. Bacteriol. 48: 1049-1055.

Order: +49(0)-421-69 61 62 0

Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 16

E. Navarro, J. Escribano, J.A. Fernndez, J. Solera (2002). Comparison of three different PCR methods for detection of Brucella spp. in human blood samples. FEMS Immunol. Med. Microbiol. 34: 147-151.

2
Kits Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation
Features of MolYsis Kits Targeted bacterial DNA isolation from blood and blood culture Human DNA removal PCR inhibitor removal Efficient lysis of Gram-positive and Gram-negative bacteria Enhancement of PCR sensiti - vity up to 20,000-fold Processing of small (0.2 ml), medium (1 ml) and large blood samples (5 ml) DNA-free reagents Bacterial DNA isolation in less than 2 hours Specimens for diagnosis of infection Whole blood samples Periodontal and caries samples Joint fluids Cerebrospinal fluids Blood cultures Blood products, including platelet and erythrocyte concentrates

17

order@molzym.com

2
MolYsis Basic and MolYsis Basic5 kits are units that are designed to treat 0.2 ml or 5 ml whole blood samples for the removal of human DNA and enrichment of bacterial cells. They can be used in conjunction with other commercial DNA Mini isolation kits, e. g. Qiagen, Roche, Promega and Epicentre products. MolYsis Basic kits can also be used with automated systems such as FuijFilm Quick Gene 810, Chemagen chemagic MSM I, Roche Magna Pure, Biomerieux easyMag or PSS Magtration. MolYsis Complete5 provides the complete solution for human DNA removal and bacterial DNA isolation from 1 ml or 5 ml whole blood and other body fluids. MolYsis Plus is the kit of choice for blood culture diagnosis. This kit relies on Molzyms technology for the removal of strong PCR inhibitors and the isolation of pure bacterial DNA.

Order: +49(0)-421-69 61 62 0

Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 18

The MolYsis kits cover the following three application catagories:

2
MolYsis Basic Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation
Human DNA removal kit for pediatric blood samples (0.2 ml)
This kit enables the removal of human DNA and enrichment of bacterial cells from small, pediatric samples of whole blood (0.2 ml). MolYsis Basic includes all the reagents for human cell lysis, human DNA cleavage and bacterial cell wall degradation as a precursor to DNA isolation. MolYsis Basic is ideally used in conjunction with commercial DNA Mini Spin column kits for the extraction of DNA from whole blood.

Applications
Sepsis diagnosis of newborn children Infection diagnosis of clinical aspirates Periodontal and caries samples

Contents: Buffers, MolDNase (DNA degrada tion), BugLysis (degradation of Gram positive and Gram-negative cell walls) Sample: 0.2 ml blood Use with: DNA Mini isolation kits, e. g. Qiagen QiaAmp Blood DNA Mini and DNeasy kits, Roche High Pure PCR Template Preparation Kit, Epicentre MasterPure DNA Purification Kit

Specifications

Storage conditions
Kit and buffers at room temperature (15-25C); enzymes (MolDNase and BugLysis) at -15C to -20C

Order information
Product MolYsis Basic Reactions 50 100 Order No. D-300-050 D-300-100

19

order@molzym.com

2
MolYsis Basic5
MolYsis Basic5 is a kit for human DNA removal and bacteria enrichment from 5 ml whole blood. This kit is used in combination with other commercial DNA Mini purification kits for the isolation of bacterial DNA and its use as a template in PCR or qPCR assays

. Contents: Buffers, MolDNase (DNA degrada tion), BugLysis (degradation of Gram positive and Gram-negative cell walls) Sample: 5.0 ml blood Use with: DNA Mini isolation kits, e. g. Qiagen QiaAmp Blood DNA Mini and DNeasy kits, Roche High Pure PCR Template Preparation Kit, Epicentre MasterPure DNA Purification Kit

Specifications

Applications
Sepsis diagnosis of adult patients Infection diagnosis of other body fluids, including cerebro spinal fluids and joint aspirates

Storage conditions
Kit and buffers at room temperature (15-25C); enzymes (MolDNase and BugLysis) at -15 C to -20C

Order information
Product MolYsis Basic5 Reactions 50 100 Order No. D-301-050 D-301-100

Order: +49(0)-421-69 61 62 0

Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 20

Human DNA removal kit for 5 ml blood samples

2
MolYsis Complete5 Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation
Human DNA removal and bacterial DNA Mini isolation kit for 5 ml and 1 ml blood samples
This kit is the complete solution for bac terial DNA isolation from whole blood. MolYsis Complete5 contains all the buffers and reagents necessary for human DNA removal, bacteria enrichment and bacterial DNA isolation from 1 ml and 5 ml whole blood samples.

Applications
Sepsis diagnosis of adult patients (5 ml protocol) Sepsis diagnosis of child patients (1 ml protocol) Infection diagnosis of other body fluids, including cerebro spinal fluids and joint aspirates

Contents: Buffers, MolDNase (DNA degrada tion), BugLysis (degradation of Gram positive and Gram-negative cell walls), Mini spin columns Sample: 5.0 ml blood (extra protocol for 1.0 ml included)

Specifications

Storage conditions
Kit and buffers at room temperature (15-25C); enzymes (MolDNase, BugLysis and proteinase K) at -15C to -20C

Order information
Product MolYsis Complete5 Reactions 50 100 Order No. D-321-050 D-321-100

21

order@molzym.com

2
MolYsis Plus Chapter 2: MolYsis - Pre-analytic Kits for Bacterial DNA Isolation 22
Bacterial DNA Mini isolation kit for blood cultures
MolYsis Plus is specially designed for the isolation of bacterial DNA from blood cultures. The kit is based on a proprietary technology for the efficient removal of strong PCR inhibitors from the medium and the isolation of extremely pure bacterial DNA for PCR and qPCR analysis.

Contents: Buffers, MolDNase (DNA degrada tion), BugLysis (degradation of Gram positive and Gram-negative cell walls), Mini spin columns Sample: 0.2 ml blood culture

Specifications

Applications
PCR/qPCR analysis of blood culture-grown pathogens

Storage conditions
Kit and buffers at room temperature (1525C); enzymes (MolDNase, BugLysis and proteinase K) at -15C to -20C

Order information
Product MolYsis Plus Reactions 50 100 Order No. D-310-050 D-310-100

Order: +49(0)-421-69 61 62 0

23

Chapter 3: MolTaq 16S and Mastermix 16S

order@molzym.com

using PCR

3
Chapter 3: MolTaq 16S and Mastermix 16S 24

Mastermix 16S Basic - Premixed PCR reagents and fluorescent dye using custom primers......................30 Mastermix 16S Basic - Premixed PCR reagents for custom assays........................................................28

Order: +49(0)-421-69 61 62 0

3
Background Information Chapter 3: MolTaq 16S and Mastermix 16S 25
The detection of bacteria in blood by PCR or Real-Time PCR (qPCR) is a growing field of interest in the clinical diagnosis of sepsis and applied testing, including sterility control of blood products. In all applications, quantitative issues are desired with a high level of detection sensitivity. There is a strong risk that DNA contamination can give rise to false positive signals in highly sensitive PCR assays targeting conserved bacterial sequences (Millar et al. 2002). This can occur during the procedure, but it can also come from the contamination of the working materials with DNA from numerous exogenous sources (Corless et al. 2000). Furthermore, a prominent source of background DNA is the PCR mastermix itself. In the first place, the polymerising enzyme can contain traces of DNA from the production strain or environmental organisms (Mhl et al. 2008). But primers and dNTPs can also be contaminated with bacterial DNA (Goto et al. 2005). The problem of DNA contamination becomes particularly problematical when bacteria are present in small numbers. Molzym has developed a technology that efficiently removes traces of DNA from PCR mastermix components. A series of DNA-free products is offered, including highly active Taq DNA polymerase and various mastermixes designed to reliably detect bacteria at highest sensitivity issues in clinical samples. Molzyms products are high purity reagents, optimised for the quantitative detection of bacteria by PCR. We are supplied with chemicals, reagents and HPLC-purified primers by our collaborating partners. Manufacturing of DNA-free Taq DNA polymerase, MolTaq 16S, and the mastermix product series, Mastermix 16S, takes place at our premises under strictly controlled sterile conditions. This also includes the use of DNA-free disposables and vessels.

Bacterial DNA-free PCR products

Molzym have introduced a series of the highest quality products for the detection of bacterial infections in blood and other body fluids using PCR which considerably reduce the problems of false-positives. Molzym offers solutions to any needs of our customers, ranging from highly active Taq DNA polymerase, MolTaq 16S, to Mastermix 16S in its various designs and an assay for the detection of bacteria, Mastermix 16S Complete. All DNA-free products enable ultra-sensitive analysis including general 16S and 23S rDNA PCRs. False-positive signals due to contaminating DNA in PCR reagents are excluded by Molzyms purification technology. The extremely high amplification activity of the PCR reagents delivers strong signals and guarantees detection of very low levels of bacterial target DNA in the sample. Strict quality control is documented with each product by filing information about the absence of DNA and the performance of the product.

order@molzym.com

3
Table 4: Overview of DNA-free PCR products

Mastermix 16S Dye

Mastermix 16S Primer Mastermix 16S Complete

Features
Bacterial DNA-free (>220 bp amplification product guarantee) Highly active Taq DNA polymerase Sensitive detection of bacteria Precise diagnosis at low bacterial target numbers Suitable for research, development and routine (non IVD) CE conforming manufacturing and validated 40 PCR cycles activity guarantee

Note:
DNA contamination in primers, probes, PCR vials, pipette tips and other disposables can cause problems with the PCR detection of bacteria. To ensure reliable amplifications, please call our team and ask for information on how to avoid DNA contamination from such sources and for the details of suppliers of DNA-free products supplementary to Mastermix 16S products. Support hotline: +49(0)421-696162-0 email: support@molzym.com References:
B. C. Millar, J. Xu, J. E. Moore (2002). Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology. J. Clin. Microbiol. 40: 1575-1580. C. E. Corless. M. Guiver, R. Borrow, V. Edwards-Jones, E. B. Kaczmarski, A. J. Fox (2000). Contamination and sensitivity issues with a Real-Time universal 16S rRNA PCR. J. Clin. Microbiol. 38: 1747-1752. M. Goto, S. Ando, Y. Hachisuka, T. Yoneyama (2005). Contamination of diverse nifH and nifH-like DNA into commercial PCR primers. FEMS Microbiol Lett. 246: 33-38. H. Mhl, A.-J. Kochem, C. Disqu, S. G. Sakka (2008) Activity and DNA contamination of commercial PCR reagents for the universal 16S rDNA Real-Time PCR detection of bacterial pathogens in blood. Diagn. Microbiol. Infect. Dis. [in press] doi:10.1016/j.diagmicrobio.2008.07.011.

Order: +49(0)-421-69 61 62 0

Chapter 3: MolTaq 16S and Mastermix 16S 26

Product MolTaq 16S Mastermix 16S Basic

Applications All PCR assays for the detection of bacteria. PCR or qPCR using custom primers PCR or qPCR using custom primers for fluorescent dye detection Included: Fluorescent dye, DNA size marker, gel loading buffer. Universal PCR or qPCR detection of bacteria. Included: Universal primers. Universal PCR or qPCR detection of bacteria using fluorescent dye. Included: Fluorescent dye, DNA size marker, gel loading buffer, universal primers.

3
MolTaq 16S Chapter 3: MolTaq 16S and Mastermix 16S
Bacterial DNA-free highly active Taq DNA polymerase
MolTaq 16S is an outstanding Taq DNA polymerase which facilitates the reliable and sensitive detection of bacteria in samples. The product is free from amplifiable DNA (>220 bp) and has an extremly high activity. MolTaq 16S is particularly useful for PCR amplifications involving primers for conserved regions of the bacterial 16S or 23S rRNA gene.

Applications
Detection of bacteria by PCR in samples Sterility control of biotechno logical, pharmaceutical and medical products Development of assays for the detection of bacteria

Contents: MolTaq 16S (5 Units/l), 10x PCR buffer and DNA-free PCR grade water PCR buffer (10x): 600 mM Tris-sulphate (pH 9.1), 180 mM ammonium sulphate, 15 mM magnesium sulphate Assay: up to 40 cycles at maximum activity

Specifications

Order information
Product MolTaq 16S Reactions 100 500 Order No. P-019-0100 P-019-0500

Features
Bacterial DNA-free 40 PCR cycles guarantee 100% active

Storage conditions
-15 to -20 C

27

order@molzym.com

3
Mastermix 16S Basic Chapter 3: MolTaq 16S and Mastermix 16S 28
Premixed PCR reagents
This mastermix includes basic components, PCR buffer, dNTPs, BSA and MolTaq 16S DNA polymerase, necessary to run amplifications under optimised reaction conditions. Mastermix 16S Basic is a 2.5x-concentrated mastermix suitable for PCRs, using custom primers, and qPCRs, using primers and fluorescent dyes or probes.

Contents: 2.5x-concentrated; PCR buffer (3 mM Mg2+ final conc.), dNTPs, BSA and MolTaq 16S, DNA-free PCR grade water included Assay: 25 l final reaction volume up to 40 cycles at maximum activity

Specifications

Applications
PCR or qPCR detection of bacteria in blood and other samples using custom primers and probes or fluorescent dyes.

Order information
Product Mastermix 16S Basic Reactions 100 250 1000 Order No. S-040-0100 S-040-0250 S-040-1000

Storage conditions
-15 to -20 C

Note: To ensure reliable amplifications, please call our team and ask for information about suppliers of DNA-free products supplementary to Mastermix 16S Basic. Support hotline: +49(0) 421-69 61 62 0 email: support@molzym.com Order: +49(0)-421-69 61 62 0

3
Mastermix 16S Dye Chapter 3: MolTaq 16S and Mastermix 16S
Premixed PCR reagents and fluorescent dye
Mastermix 16S Dye is a mastermix intended for the detection of bacterial DNA by PCR and agarose gel electrophoresis or qPCR using custom validated primers and fluorescent dye. It includes PCR buffer, dNTPs, BSA, MolTaq 16S DNA polymerase and a fluorescent dye.

Applications
PCR or qPCR detection by fluorescent dye of bacteria or fungi in blood and other samples using custom primers.

Contents: 2.5x-concentrated; PCR buffer (3 mM Mg2+ final conc.), dNTPs, BSA, MolTaq 16S and fluorescent dye, DNA-free PCR-grade water Assay: 25 l final reaction volume up to 40 cycles at maximum activity

Specifications

Order information
Product Mastermix 16S Dye Reactions 100 250 1000 Order No. S-030-0100 S-030-0250 S-030-1000

Storage conditions
-15 to -20 C

Note: To ensure reliable amplifications, please call our team and ask for information about suppliers of DNA-free products supplementary to Mastermix Dye. Support hotline: +49(0) 421-69 61 62 0 email: support@molzym.com

29

order@molzym.com

3
Mastermix 16S Primer Chapter 3: MolTaq 16S and Mastermix 16S 30
Premixed PCR reagents and universal 16S rDNA primers
Mastermix 16S Primer includes primers which bind to conserved regions of the 16S rRNA gene. This mastermix is particularly useful for the sensitive detection of bacteria using PCR followed by sequencing of the amplification product and strain identification by sequence library search (www.sepsitest-blast.net). Mastermix 16S Primer can also be used for qPCRs using fluorescent dye or probe detection.

Contents: 2.5x-concentrated; PCR buffer (3 mM Mg2+ final conc.), dNTPs, BSA, MolTaq 16S and universal 16S rDNA primers. DNA-free, PCR-grade water. Ask for sequencing primers! Assay: 25 l final reaction volume up to 40 cycles at maximum activity

Specifications

Applications
PCR detection and identifi cation by sequencing of bacteria in blood and other samples by amplification using universal 16S rDNA primers.

Order information
Product Reactions 100 Order No. S-021-0100 S-021-0250 S-021-1000

Storage conditions
-15 to -20 C

Mastermix 16S Primer

250 1000

Note: To ensure reliable amplifications, please call our team and ask for information about suppliers of DNA-free products supplementary to Mastermix 16S Primer. Support hotline: +49(0) 421-69 61 62 0 email: support@molzym.com Order: +49(0)-421-69 61 62 0

3
Chapter 3: MolTaq 16S and Mastermix 16S Mastermix 16S Complete
Universal 16S rDNA assay for fluorescent dye detection of bacteria

Mastermix 16S Complete is a DNA-free master mix especially designed as an assay of bacteria (Gram-positives and Gram-negatives) by PCR and gel electrophoretic analysis or qPCR using universal 16S rDNA primers and a fluorescent dye. This mastermix contains all the ingredients necessary to run the assay after adding the template. Mastermix 16S Complete is thus the best way to exclude reagent-borne DNA contamination.

Applications
PCR or qPCR assay for the universal detection (16S rDNA) of bacteria in blood and other samples. Specifications
Contents: 2.5x-concentrated; PCR buffer (3 mM Mg2+ final conc.), dNTPs, BSA, MolTaq 16S, fluorescent dye and universal 16S rDNA primers, DNA-free PCR grade water, DNA size marker, gel loading buffer. Ask for sequencing primers! Assay: 25 l final reaction volume up to 40 cycles at maximum activity

Order information
Product Mastermix 16S Complete Reactions 100 250 1000 Order No. S-020-0100 S-020-0250 S-020-1000

Note: To ensure reliable amplifications, please call our team and ask for information about suppliers of DNA-free products supplementary to Mastermix 16S Complete. Support hotline: +49(0) 421-69 61 62 0 email: support@molzym.com

Storage conditions
-15 to -20 C

31

order@molzym.com

3
Chapter 3: MolTaq 16S and Mastermix 16S
Note: To ensure reliable amplifications, please call our team and ask for information about suppliers of DNA-free products supplementary to customised Mastermix 16S. Support hotline: +49(0) 421-69 61 62 0 email: support@molzym.com Order: +49(0)-421-69 61 62 0

DNA decontamination service for customised mastermix

You have developed a qPCR assay (not hotstart) to detect bacteria and you face problems with false positive signals from DNA contamination emanating from the mastermix? Molzyms service comprises the assembly and purification of qPCR components, including PCR buffer (3 mM Mg2+ final concentration), dNTPs, BSA, MolTaq 16S and custom specified primers, producing a mastermix that allows runs of quantitative assays at the specific conditions laid down by the customer without false positives being produced by traces of DNA in the reagents. A guarantee for the absence of DNA contamination is given for PCR primers resulting in >220 bp amplification product. A detailed certificate of analysis is supplied with the DNA-free mastermix.

Contents: 2.5x-concentrated; PCR buffer (3 mM Mg2+ final conc.), dNTPs, BSA, MolTaq 16S, fluorescent dye and custom primers, DNA-free PCR grade water.

Specifications

Assay: 25 l final reaction volume up to 40 cycles at maximum activity

32

4
Chapter 4: SepsiTest - Pathogen detection kit

CE IVD Pathogen Detection Kit

33

order@molzym.com

4
Chapter 5: SepsiTest - Pathogen detection kit 34

- Complete CE IVD System isolation and universal rDNA PCR............................36

Order: +49(0)-421-69 61 62 0

4
Introduction to the System Chapter 4: SepsiTest - Pathogen detection kit
SepsiTest is a kit for the in vitro diagnosis of blood stream infections (CE-IVD marked). SepsiTest combines pre-analytic and diagnostic solutions forming a unit for the detection and monitoring of bacteria and yeasts in whole blood. The kit relies on Molzyms innovative technology of human DNA-free isolation of bacterial and yeast target DNA from whole blood and universal rDNA assays to provide a high quality, straight forward kit for molecular detection of pathogens. SepsiTest supplies two separate assays with primers targeting eubacterial and Candida spp. rDNA sequences, respectively. Detection can be by PCR and agarose gel electrophoretic analysis using a fluorescent dye. Real-Time PCR (qPCR) is a researchuse-only option. SepsiTest can be easily followed by sequencing of amplification products. An online BLAST tool (www. sepsitest-blast.net) can be used for strain identification. Primers and a protocol necessary for sequencing analysis are included in the kit. In a prospective study including 187 critically ill patients from 4 hospitals with signs of SIRS, sepsis, neutropenia or immunodeficiency, blood samples were examined for pathogens using SepsiTest and blood culture (BC). In the case of distinct PCR signals coming up in the SepsiTest test, amplification products were sequenced and strains identified by BLAST analysis (www.sepsitest-blast.net).
Table 5: Study of blood samples from SIRS and neutropenic patients for bacterial pathogens

Blood culture PCR + sequencing Positive Negative Total Positivity (%): Blood culture PCR + sequencing Sensitivity (%) Specificity (%) PPV (%) NPV (%) positive 47 7 54 negative 41 247 288 15.8 25.7 87.0 85.8 53.4 97.2 total 88 254 342

* PPV, positive predictive value, NPV, negative predictive value; Disqu, personal communication

Among the 342 samples, 54 BCs (34 patients) were positive, of which 47 (28 patients) were also PCR+sequencing-positive. The resulting sensitivity and specificity values were high (Table 5). Positive PCR+sequencing results were further obtained with 41 BC-negative samples from 31 patients (Table 5), resulting in a positivity rate 1.6 times higher than BC. Taking microbiological data from other body sites and clinical aspects including antibiotic treatment into account, PCR results indicated that among the BC-negative patients 24 (77%) were likely to be infected. The data show that SepsiTest together with sequencing is a reliable method for the detection and identification of pathogens in blood samples. With the time needed from sample preparation to pathogen detection and sequencing analysis in only approx. 6-7 hours, is a means of rapid diagnosis that can accompany standard microbiological testing.

35

order@molzym.com

4
SepsiTest CE IVD Chapter 4: SepsiTest - Pathogen detection kit 36
Complete system of targeted bacterial and yeast DNA isolation and universal rDNA PCR assays
This kit constitutes a combination of pre-analytic and diagnostic units for the detection of pathogens in whole blood. SepsiTest combines human DNA-free microbial DNA isolation and universal rDNA PCR detection of pathogens in septicemic blood samples of child and adult patients. Sequencing of the amplification product is an option for the identification of pathogens following the detection by SepsiTest.

Contents: A) DNA Isolation Unit: reagents for human DNA removal and spin column isolation of bacterial and yeast DNA from 1 ml whole blood (duplicate extraction) B) Detection assay unit: 2.5x-concentrated mastermix (25 l max. final reaction volume); PCR buffer (3 mM Mg2+ final conc.), dNTPs, BSA, MolTaq 16S, fluorescent dye, universal rDNA primers, sequencing primers (10 M) for the identification for bacteria and yeasts. Assay 1: bacteria (CE IVD) Assay 2: yeasts Assay 3: internal positive PCR control (inhibition control)

Specifications

Applications
Routine diagnosis of sepsis by universal bacteria and yeast detection and sequencing analysis. CE-marked for in vitro diagnostics (98/97 EC)

Order information
Product SepsiTest Samples 12 24 Order No. A-020-024 A-020-048

Storage condition
DNA isolation unit (buffers, spin columns): room temperature DNA isolation enzymes and detection assay unit: -15 to -20 C

Note: Please call our team and ask for information about suppliers of DNA-free products supplementary to SepsiTest for reliable results. Support hotline: +49(0) 421-69 61 62 0 email: support@molzym.com

Order: +49(0)-421-69 61 62 0

Terms and Conditions

The sale of goods of Molzym GmbH & Co. KG (hereinafter referred to as the Seller) to entrepreneurs within the meaning of 14 German Civil Code (Brgerliches Gesetzbuch, BGB) shall exclusively be governed by the General Terms and Conditions of Molzym GmbH & Co. KG in the version respectively in effect at the time of the conclusion of each contract. Any contradicting or deviating general terms and conditions of the Buyer shall not become part of the contract unless the Seller has expressly agreed to their applicability in writing. This shall also apply in the case that the Seller, with knowledge of contradictory or deviating terms and conditions of the Buyer, performs the contract without making a statement of reservation. In any case, the Buyers consent to these General Terms and Conditions shall be deemed given, when the Buyer breaks the seal on the outer packaging of the Sellers goods, unless it returns the goods with untouched inner packaging within ten (10) business days following the breaking of the seal, however not later than twenty (20) days following shipment, to the Seller for a full refund. The Seller undertakes to inform the Buyer of this effect of opening the outer packaging with a clearly visible seal on the outer packaging. 1. Sellers offers are subject to change without notice. The Seller shall reserve the right to make minor deviations from its specifications concerning dimensions, weight, condition and quality. 2. a) Delivery dates shall be approximate, unless the Seller has recognized such in writing to be binding. b) Should Seller fail to meet a stipulated delivery date, Buyer may only after unsuccessful expiration of a reasonable period of grace set by it rescind the contract or claim for damages instead of the performance (Schadensersatz statt der Leistung). This does not apply insofar, as Seller is responsible for his failure to meet the delivery date, or the setting of a period of grace is dispensable pursuant to 323 para. 2 or 281 para. 2 of the German civil code (BGB). In case of a partial fulfill ment by the Seller, the Buyer shall only be entitled to rescind the entire contract (Rcktritt vom ganzen Vertrag), if it has no interest in the performance taking into account an objective standard. 3. Only those units listed in the Sellers currently valid price lists shall be deliverable. Seller shall be authorized to make deliveries in installments. Each installment may be invoiced separately. With orders deliverable on call, notice thereof must be made at least two weeks prior to the designated delivery date. 4. Force majeure, company shutdowns, labor disputes or other impediments which are outside the Sellers responsibility which affect the Seller or its suppliers shall release the Seller from the contractual delivery obligations for the term of the disruption and its effects. 5. a) Seller shall determine the type and manner of shipping, insofar as not otherwise instructed in writing by the Buyer. b) Shipping shall be made ex works Bremen, insofar as not otherwise agreed. Orders will be charged a shipping fee of 20 (Germany). Products that require shipping on dry ice will be charged an additional fee of 20 (Germany). c) Buyer shall bear the risk of incidental loss or incidental deterioration of the goods shipped as soon as the Seller hands over the goods to the shipping carrier. 6. a) Pricing is in EURO. Prices shall include the packaging costs. Value added tax shall be added thereon. Buyer shall bear the shipment costs, insofar as not otherwise agreed. b) Should Seller, after expiration of four months from the date of the conclusion of the sales contract, i.e. usually after Sellers order confirmation, generally increase or reduce its prices, then the prices in effect on the delivery date shall apply. 7. a) Sellers invoices shall be payable without deductions within 14 days from the date of delivery. b) Bills of exchange shall not be accepted as a means of payment. Checks shall only be accepted pending full discharge of the debt. c) In the event of late payment, Seller shall assess interest as of the due date, without a dunning notice, in the amount of 8 percent points above the base interest rate within the meaning of 247 BGB. d) Buyer may only set-off its own claims against due payments or claim a right of retention insofar as its claims are determi ned with res judicata effect, are non-disputed or are recognized. In addition, Buyer shall not be permitted to assign its claims against Seller. 8. a) Seller reserves ownership title to the goods delivered by it until the Buyer has discharged all of its obligations arising out of the business relationship with Seller. The goods subject to reservation of title may neither be pledged nor transferred as security. Buyer shall only be authorized to sell the goods subject to the reservation of title in the ordinary course of its business. b) To secure Sellers claims from the business relationship with the Buyer, Buyer herewith now assigns to the Seller a first priority creditor right to its accounts receivable resulting from the resale of the goods subject to reservation of title in the amount of the Sellers invoice. Payments which the Buyer receives as payment for the sale of goods subject to reservation of title shall first be credited to that part of the total accounts receivable not assigned to the Seller, insofar as the payer does not expressly state otherwise. c) Insofar as reservations of title in the Sellers favor exist or accounts receivable of the Buyer are assigned to the Seller, then the Buyer shall be obligated to provide any information necessary for the protection of the Sellers rights. This shall apply, in particular, to attachments or other forms of seizure or arrest by third parties on the goods or any accounts receivable assigned to the Seller. The costs of any interventions shall be borne by the Buyer. d) Subject to revocation of such right, the Buyer shall be authorized to collect the accounts receivable assigned to the Seller. The Sellers right to collect the assigned accounts receivable itself shall remain unaffected hereby. e) Insofar as the value of the security granted exceeds the amount of the Sellers claims by more than 20%, the Seller shall be obligated to re-assign the security in the respective amount. f) Upon the full performance of Sellers claims, including all auxiliary claims, the respective security shall be automatically transferred back to the Buyer without a special transfer action. 9. a) The purchase of the goods only conveys to Buyer the license to use the purchased goods according to the product manual and subject to the following license conditions. b) Sellers goods are designed exclusively for use in scientific research and development. Any use of the Sellers goods for human medical treatment, for in vitro diagnostic purposes, or as pharmaceuticals shall not be permitted.

Terms and Conditions 37

order@molzym.com

c) Any copying, disassembling, analyzing, sequencing or other form of reverse engineering of Sellers goods is expressly forbidden. d) Any resale of the goods delivered by the Seller is only permitted if the Buyer agrees with its customer to bind the customer to terms identical to this item 9 a) - e) in favor of the Seller. e) THE BUYER HEREBY ASSIGNS ANY RIGHTS, INCLUDING, BUT NOT LIMITED TO PATENTS AND PATENT APPLICATIONS, THAT IT MAY OBTAIN DUE TO A VIOLATION OF ITS OBLIGATIONS UNDER ITEMS 9a), b), c) OR d) TO THE SELLER, WHICH ACCEPTS SUCH ASSIGNMENT. FURTHERMORE, THE BUYER SHALL PAY TO THE SELLER A ROYALTY IN THE AMOUNT OF 30% OF ALL NET SALES OF PRODUCTS DEVELOPED DUE TO SUCH VIOLATION. FURTHERMORE, THE BUYER SHALL INDEMNIFY AND HOLD HARMLESS THE SELLER OF ANY THIRD PARTIES CLAIMS BASED ON SUCH VIOLATION, AND SHALL BE LIABLE FOR ANY DAMAGES OF THE SELLER INSOFAR AS THEY HAVE NOT BEEN CURED BY THE AFORESAID PROVISIONS. 10. a) Notifications of defects of goods delivered or deviations of quantity or incorrect deliveries shall be made in writing at the latest within one week after receipt of the goods. Latent defects shall be notified without undue delay after their discovery. The failure to observe these deadlines shall result in the automatic loss of any warranty claims which might otherwise have existed. b) In case of justified objections, the Seller shall within a reasonable period supply the missing quantities, or, at Sellers discretion, replace the goods or rectify the defect. c) Should the Buyer have set a reasonable period of grace for subsequent performance within the meaning of Item 10 b), then the Buyer can, after unsuccessful expiration of the period set by it, demand either a reduction of the purchase price or rescind the contract. The requirement of the setting of a reasonable period of grace does not apply insofar as the setting of a period of grace is dispensable pursuant to 323 para. 2 BGB, the subsequent performance failed, is unacceptable for the Buyer or has been refused by the Seller. In case of delivery of defective goods, the Buyer shall only be entitled to rescind the contract if it has no interest in the performance taking into account an objective standard. d) The Seller shall be liable in accordance with the statutory provisions for damages which were caused by intentional misconduct or gross negligence of the Sellers legal representatives or management employees, for fraudulently non disclosed defects, for personal damages, for claims pursuant to the German Product Liability Act, for initial impossibility insofar as the Seller had known or should have known of the initial impossibility at the time of the conclusion of the contract, for its performance of its essential obligations, and for stipulated attributes of the goods sold, insofar as the Seller assumed a guarantee for their attributes. The Seller shall be liable for damages in the amount of the typical and foreseeable losses resulting from negligent violations of Sellers essential contractual obligations or fundamental obliga tions and for damages caused by Sellers employees as a result of gross negligence or intention without violating essential contractual provisions or fundamental obligations. In case of a partial performance or the delivery of defective goods, the Buyer shall be entitled to damages instead of the entire performance (Schadensersatz statt der ganzen Leistung) only if it has no interest in the performance taking into account an objective standard. Otherwise, any liability shall be excluded. e) No warranty claims or damage claims shall be allowed in the event of inappropriate handling and processing of the Sellers goods. f) The limitation period for claims of the Buyer resulting from defects shall be one year following delivery of the goods. This deadline shall also apply for claims based on tort resulting from defects of the goods. Should the Buyer be in default of acceptance, then the limitation period shall start to run upon the transfer of risk. Claims of the Buyer other than claims based on defects, in particular, claims on the basis of accessory obligations, pre-contractual liability or tort shall be time barred two years after delivery of the goods. The afore mentioned limitation periods shall not apply to claims of the Buyer pursuant to Item 10 d) hereof to which it is entitled on the basis of the same facts. 11. a) Place of performance and payment shall be Bremen. For Buyers who are business persons or who have their domicile outside of the Federal Republic of Germany, jurisdiction shall be with the Local Court in Bremen or, as the case may be (for disputes concerning claims with a value in excess of Euro 5,000.), the District Court in Bremen. The Seller may, however, elect to have such disputes decided by the courts having jurisdiction at the domicile of the Buyer. b) German law shall apply. The UN Convention on Contracts for the International Sale of Goods (CISG) shall not be applicable.

-69 61 Order: +49(0)-421-22 09 62 77 0 70

Terms and Conditions 38

Customer Service
Ordering Information
To order please use any of the following options:

Postal: Phone: Fax: Email:

Molzym GmbH & Co. KG Mary-Astell-Strasse 10 28359 Bremen Germany +49(0)421-69 61 62 0 +49(0)421-69 61 62 11 order@molzym.com

Technical Service
Molzyms team of Technical Service Representatives rely on years of laboratory experience to respond to our customers technical questions and assist with product selection. You may contact Technical Service by phone or e-mail: +49(0)421-69 61 62 0 support@molzym.com