Technology
Pathogen DNA Enrichment and Universal Detection/Identification for in-vitro Diagnostics
Several kits are available for the pre-analytic treatment of and pathogen DNA isolation from such diverse specimens like blood, CSF, BAL, synovial and pleural aspirates, swabs, tissues and biopsies. All the specimens contain human DNA which generally is in large excess to pathogen DNA. In unspecific reactions, PCR primers directed to conserved regions of the 16S and 18S rRNA genes, respectively, can interfere with human DNA during the PCR amplification reaction in a way that false-positive results may arise. In addition, primers can be diluted by the unspecific binding to human DNA with the result of false negative results. Molzym’s patented technology of pre-analytic removal of human DNA from clinical specimens, MolYsis, enables the enrichment of bacterial and fungal DNA and hence the increase in detection sensitivity of pathogens (click here for more detailed information).
Molzym’s kits, SepsiTest™, UMD™ and automated SepsiTest™ SelectNA rely on the universal 16S and 18S rRNA gene-targeted PCR or Real-Time PCR amplification and sequencing identification. The regions amplified contain highly variable sequences allowing identification of the pathogens to the species level. The universal nature of the PCR reaction allows the detection and identification of any pathogen present in a sample. Molzym has developed an online tool, SepsiTest™ BLAST which displays the identification results in a plain way. All components of the kits are manufactured under strict quality control thereby guaranteeing absence of contaminating microbial DNA in the reagents and consumables supplied with the kits.
Molzym’s kits, SepsiTest™, UMD™ and automated SepsiTest™ SelectNA rely on the universal 16S and 18S rRNA gene-targeted PCR or Real-Time PCR amplification and sequencing identification. The regions amplified contain highly variable sequences allowing identification of the pathogens to the species level. The universal nature of the PCR reaction allows the detection and identification of any pathogen present in a sample. Molzym has developed an online tool, SepsiTest™ BLAST which displays the identification results in a plain way. All components of the kits are manufactured under strict quality control thereby guaranteeing absence of contaminating microbial DNA in the reagents and consumables supplied with the kits.
Automated Solution for in-vitro Diagnostics
SepsiTest™ SelectNA stands for simplicity and reliance in molecular analysis of blood samples for etiological agents of sepsis. The automated part, SelectNA™, comprises of a sophisticated, high precision instrument specially designed for the isolation of DNA from a broad-range of organisms. SelectNA™ guarantees a contained environment avoiding air-borne immissions of bacterial DNA during processing samples. The strong UV source moves in the interior thereby inactivating microbes and DNA.
The kit delivered, SepsiTest™ SelectNA enables the automated extraction and purification of DNA from pathogens enriched from blood by the sample pre-treatment. Subsequently, the eluate is included in supplied PCR or Real-Time PCR assays for the universal detection of bacteria and fungi. First results of the infectious state of patients (presence/absence of bacteria and/or fungi) are gained after approximately 4h with only 75 min hands-on time (4 samples).
The kit delivered, SepsiTest™ SelectNA enables the automated extraction and purification of DNA from pathogens enriched from blood by the sample pre-treatment. Subsequently, the eluate is included in supplied PCR or Real-Time PCR assays for the universal detection of bacteria and fungi. First results of the infectious state of patients (presence/absence of bacteria and/or fungi) are gained after approximately 4h with only 75 min hands-on time (4 samples).
Sequencing analysis of positive samples is the method of identifying an organism. Sequencing primers are supplied with each kit. This process follows usual standards of operation with Sanger-directed sequence analysis systems. The total processing time is approx. 260 min with 70 min hands-on time. Typically, sequencing reactions are performed over night with next morning identification results available.
Clinical evaluation
Molzym is continuously performing clinical studies for the evaluation of SepsiTest™, UMD™ and SepsiTest™ SelectNA kits. Molzym’s efforts are directed to comparing the kits’ performance with standard microbiological culturing methods. All kits are marked for the in-vitro diagnosis of infectious agents (98/79 EC).
Click here to download a list of organisms detectable, including representatives of genera identified by sequencing in the studies so far.
Latest number of species detectable:
Gram-negative (169)
Gram-positive (179)
Fungi (20)
Date: October, 2011
Click here to download a list of organisms detectable, including representatives of genera identified by sequencing in the studies so far.
Latest number of species detectable:
Gram-negative (169)
Gram-positive (179)
Fungi (20)
Date: October, 2011
References
Kühn C, Disqué C, Mühl H, Orszag P, Stiesch M, Haverich A (2011) Evaluation of commercial universal rRNA gene PCR plus sequencing tests for identification of bacteria and fungi associated with infectious endocarditis. J Clin Microbiol 49, 2919-2923.
Schaub N, Frei R, Müller C (2011) Addressing unmet clinical needs in the early diagnosis of sepsis. Swiss Med Wkly. 141 (in press); doi:10.4414/smw.2011.13244
Pletz MW, Wellinghausen N, Welte T (2011) Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients? A clinical view. Intensive Care Med 37, 1069–1076.
Oliva A, Belvisi V, Iannetta M, Andreoni C, Mascellino MT, Miriam Lichtner M, Vullo V, Mastroianni CM (2010) Pacemaker lead endocarditis due to multidrug-resistant Corynebacterium striatum detected with sonication of the device. J Clin Microbiol 48, 4669-4671.
Nieman AE, Rozemeijer W, de Jong E, Beishuizen B, van Agtmael MA, Savelkoul PHM, Schade RP (2010) Molecular detection of bacterial bloodstream infections: validation of the SepsiTest™ assay. Ned Tijdschr Med Microbiol 18 Supplement, Abstract P048, 109.
von Loewenich FD, Geißdörfer W, Disqué C, Matten J, Schett G, Sakka SG, Bogdan C (2010) Detection of "Candidatus Neoehrlichia mikurensis" in two patients with severe febrile illnesses: evidence for a european sequence variant. J Clin Microbiol 48, 2630-2635.
Disqué C, Sakka SG, Wellinghausen N (2010) Klinische Evaluation von SepsiTest™ zum universellen PCR-Nachweis von bakteriellen Erregern in Vollblut. Der Mikrobiologe Heft 1, Februar 2010, 13-16.
Sakka SG, Kochem AJ, Disqué C, Wellinghausen N (2009) Blood infection diagnosis by 16S rDNA broad-spectrum polymerase chain reaction: the relationship between antibiotic treatment and bacterial DNA load. Anesthesia Analgesia 109, 1707-1708.
Wellinghausen N, Kochem AJ, Disqué C, Mühl H, Gebert S, Winter J, Matten J, Sakka SG (2009) Diagnosis of bacteremia in whole-blood samples by use of a commercial universal 16S rRNA gene-based PCR and sequence analysis. J Clin Microbiol 47, 2759-2765.
Schaub N, Frei R, Müller C (2011) Addressing unmet clinical needs in the early diagnosis of sepsis. Swiss Med Wkly. 141 (in press); doi:10.4414/smw.2011.13244
Pletz MW, Wellinghausen N, Welte T (2011) Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients? A clinical view. Intensive Care Med 37, 1069–1076.
Oliva A, Belvisi V, Iannetta M, Andreoni C, Mascellino MT, Miriam Lichtner M, Vullo V, Mastroianni CM (2010) Pacemaker lead endocarditis due to multidrug-resistant Corynebacterium striatum detected with sonication of the device. J Clin Microbiol 48, 4669-4671.
Nieman AE, Rozemeijer W, de Jong E, Beishuizen B, van Agtmael MA, Savelkoul PHM, Schade RP (2010) Molecular detection of bacterial bloodstream infections: validation of the SepsiTest™ assay. Ned Tijdschr Med Microbiol 18 Supplement, Abstract P048, 109.
von Loewenich FD, Geißdörfer W, Disqué C, Matten J, Schett G, Sakka SG, Bogdan C (2010) Detection of "Candidatus Neoehrlichia mikurensis" in two patients with severe febrile illnesses: evidence for a european sequence variant. J Clin Microbiol 48, 2630-2635.
Disqué C, Sakka SG, Wellinghausen N (2010) Klinische Evaluation von SepsiTest™ zum universellen PCR-Nachweis von bakteriellen Erregern in Vollblut. Der Mikrobiologe Heft 1, Februar 2010, 13-16.
Sakka SG, Kochem AJ, Disqué C, Wellinghausen N (2009) Blood infection diagnosis by 16S rDNA broad-spectrum polymerase chain reaction: the relationship between antibiotic treatment and bacterial DNA load. Anesthesia Analgesia 109, 1707-1708.
Wellinghausen N, Kochem AJ, Disqué C, Mühl H, Gebert S, Winter J, Matten J, Sakka SG (2009) Diagnosis of bacteremia in whole-blood samples by use of a commercial universal 16S rRNA gene-based PCR and sequence analysis. J Clin Microbiol 47, 2759-2765.