First molecular determination of herpesvirus from two mysticete species stranded in the Mediterranean Sea

BACKGROUND Herpesvirus can infect a wide range of animal species: mammals, birds, reptiles, fish, amphibians and bivalves. In marine mammals, several alpha- and gammaherpesvirus have been identified in some cetaceans and pinnipeds species. To date, however, this virus has not been detected in any member of the Balaenoptera genus. CASE PRESENTATION Herpesvirus was determined by molecular methods in tissue samples from a male fin whale juvenile (Balaenoptera physalus) and a female common minke whale calf (Balaenoptera acutorostrata) stranded on the Mediterranean coast of the Region of Valencia (Spain). Samples of skin and penile mucosa from the fin whale and samples of skin, muscle and central nervous system tissue from the common minke whale tested positive for herpesvirus based on sequences of the DNA polymerase gene. Sequences from fin whale were identical and belonged to the Alphaherpesvirinae subfamily. Only members of the Gammaherpesvirinae subfamily were amplified from the common minke whale, and sequences from the muscle and central nervous system were identical. Sequences in GenBank most closely related to these novel sequences were viruses isolated from other cetacean species, consistent with previous observations that herpesviruses show similar phylogenetic branching as their hosts. CONCLUSIONS To our knowledge, this is the first molecular determination of herpesvirus in the Balaenoptera genus. It shows that herpesvirus should be included in virological evaluation of these animals.

Fixing, storage time and DNA extraction applied to dT-RFLP for ecological and molecular studies of marine nematodes assemblages

The application of molecular methods offers an alternative faster than traditional methods based on morphology It is nearly impossible to process all the samples in short period using traditional methods, and the deterioration of marine sediments rapidly occurs The dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows a rapid assessment of biodiversity changes of nematodes assemblages The use of a not suitable fixing, storage time and DNA extraction could be a limitation in molecular analysis like dT-RFLP and real time PCR.Objetives: the best fixative •the level of DNA degradation over the time •the best DNA extraction method for marine nematodes and suitable for dT-RFLP analysis

Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits

Molecular methods are fundamental tools for the diagnosis of viral infections. While interpretation of results is straightforward for unvaccinated animals, where positivity represents ongoing or past infections, the presence of vaccine virus in the tissues of recently vaccinated animals may mislead diagnosis. In this study, we investigated the interference of RHDV2 vaccination in the results of a RT-qPCR for RHDV2 detection, and possible associations between mean Cq values of five animal groups differing in age, vaccination status and origin (domestic/wild). Viral sequences from vaccinated rabbits that died of RHDV2 infection (n = 14) were compared with the sequences from the commercial vaccines used in those animals. Group Cq means were compared through Independent t-test and One-way ANOVA. We proved that RHDV2 vaccine-RNA is not detected by the RT-qPCR as early as 15 days post- vaccination, an important fact in assisting results interpretation for diagnosis. Cq values of vaccinated and non-vaccinated infected domestic adults showed a statistically significant difference (p < 0.05), demonstrating that vaccination-induced immunity reduces viral loads and delays disease progression. Contrarily, in vaccinated young rabbits higher viral loads were registered compared to non-vaccinated kittens. No significant variation (p = 0.3824) was observed between viral loads of non- vaccinated domestic and wild RHDV2-victimised rabbits. Although the reduced number of vaccinated young animals analysed hampered a robust statistical analysis, this occurrence suggests that passively acquired maternal antibodies may inhibit the active immune response to vaccination, delaying protection and favouring disease progression. Our finding emphasises the importance of adapting kitten RHDV2 vaccination schedules to circumvent this interference phenomenon.

An investigation of Staphylococcus aureus and related species from flood affected and other environmental sources

This research investigated the microbial air quality of flooded houses in Brisbane suburbs following the January 2011 flood event. Flood waters can carry and spread human pathogenic bacteria, and these organisms can be dispersed into residential air by aerosolisation. This study found that the bacterial load was significantly different for indoor and outdoor areas of flood affected houses, but no significant differences were observed between flooded and non-flooded houses. This could be due to the rapid clean-up of flooded houses following the event. Molecular methods were used to identify and characterise staphylococcal species in residential air of flooded and non-flooded houses. A major finding was the diverse population of airborne staphylococci as well as the high rate of methicillin-resistance in these strains. By determining the genetic relatedness of residential air sourced staphylococci, a potential source for pathogenic strains can be identified.

Leptospirosis from water sources

Leptospirosis outbreaks have been associated with many common water events including water consumption, water sports, environmental disasters and occupational exposure. The ability of leptospires to survive in moist environments makes them a high risk agent for infection following contact with any contaminated water source. Water treatment processes reduce the likelihood of leptospirosis or other microbial agents causing infection provided they do not malfunction and the distribution networks are maintained. Notably, there are many differences in water treatment systems around the world, particularly between developing and developed countries. Detection of leptospirosis in water samples is uncommonly performed by molecular methods.

Prevalence of methicillin resistance and virulence determinants of Staphylococcus aureus in diabetic foot ulcers

Background Diabetic foot ulceration (DFU) is a multifactorial process and is responsible for considerable morbidity and contributes to the increasing cost of health care worldwide. The diagnosis and identification of these ulcers remains a complex problem. Bacterial infection is promoted in the diabetic foot wound by decreased vascular supply and impaired host immune response. As conventional clinical microbiological methods are time-consuming and only identifies about 1% of the wound microbiota, detection of bacteria present in DFUs using molecular methods is highly advantageous and efficient. The aim of this study was to assess the virulence and methicillin resistance profiles of Staphylococcus aureus detected in DFUs using DNA-based methods. Methods A total of 223 swab samples were collected from 30 patients from March to October 2012. Bacterial DNA was extracted from the swab samples using standard procedures and was used to perform polymerase chain reaction (PCR) using specific oligonucleotide primers. The products were visualized using agarose gel electrophoresis. Results S. aureus was detected in 44.8% of samples. 25% of the S. aureus was methicillin-resistant S. aureus harboring the mecA gene. The alpha-toxin gene was present in 85% of the S. aureus positive samples. 61% of the S. aureus present in DFU samples harbored the exfoliatin factor A gene. Both the fibronectin factor A and fibronectin factor B gene were detected in 71% and 74% of the S. aureus positive samples. Conclusions DNA-based detection and characterization of bacteria in DFUs are rapid and efficient and can assist in accurate, targeted antibiotic therapy of DFU infections. The majority of S. aureus detected in this study were highly virulent and also resistant to methicillin. Further studies are required to understand the role of S. aureus in DFU trajectory.

A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

Trapping to monitor tephritid movement: Results, best practice, and assessment of alternatives

Movement of tephritid flies underpins their survival, reproduction, and ability to establish in new areas and is thus of importance when designing effective management strategies. Much of the knowledge currently available on tephritid movement throughout landscapes comes from the use of direct or indirect methods that rely on the trapping of individuals. Here, we review published experimental designs and methods from mark-release-recapture (MRR) studies, as well as other methods, that have been used to estimate movement of the four major tephritid pest genera (Bactrocera, Ceratitis, Anastrepha, and Rhagoletis). In doing so, we aim to illustrate the theoretical and practical considerations needed to study tephritid movement. MRR studies make use of traps to directly estimate the distance that tephritid species can move within a generation and to evaluate the ecological and physiological factors that influence dispersal patterns. MRR studies, however, require careful planning to ensure that the results obtained are not biased by the methods employed, including marking methods, trap properties, trap spacing, and spatial extent of the trapping array. Despite these obstacles, MRR remains a powerful tool for determining tephritid movement, with data particularly required for understudied species that affect developing countries. To ensure that future MRR studies are successful, we suggest that site selection be carefully considered and sufficient resources be allocated to achieve optimal spacing and placement of traps in line with the stated aims of each study. An alternative to MRR is to make use of indirect methods for determining movement, or more correctly, gene flow, which have become widely available with the development of molecular tools. Key to these methods is the trapping and sequencing of a suitable number of individuals to represent the genetic diversity of the sampled population and investigate population structuring using nuclear genomic markers or non-recombinant mitochondrial DNA markers. Microsatellites are currently the preferred marker for detecting recent population displacement and provide genetic information that may be used in assignment tests for the direct determination of contemporary movement. Neither MRR nor molecular methods, however, are able to monitor fine-scale movements of individual flies. Recent developments in the miniaturization of electronics offer the tantalising possibility to track individual movements of insects using harmonic radar. Computer vision and radio frequency identification tags may also permit the tracking of fine-scale movements by tephritid flies by automated resampling, although these methods come with the same problems as traditional traps used in MRR studies. Although all methods described in this chapter have limitations, a better understanding of tephritid movement far outweighs the drawbacks of the individual methods because of the need for this information to manage tephritid populations.

Bovine cysticercosis-Development of a real-time PCR to enhance classification of suspect cysts identified at meat inspection

Laboratory confirmation methods are important in bovine cysticerosis diagnosis as other pathologies can result in morphologically similar lesions resulting in false identifications. We developed a probe-based real-time PCR assay to identify Taenia saginata in suspect cysts encountered at meat inspection and compared its use with the traditional method of identification, histology, as well as a published nested PCR. The assay simultaneously detects T. saginata DNA and a bovine internal control using the cytochrome c oxidase subunit 1 gene of each species and shows specificity against parasites causing lesions morphologically similar to those of T. saginata. The assay was sufficiently sensitive to detect 1 fg (Ct 35.09 +/- 0.95) of target DNA using serially-diluted plasmid DNA in reactions spiked with bovine DNA as well as in all viable and caseated positive control cysts. A loss in PCR sensitivity was observed with increasing cyst degeneration as seen in other molecular methods. In comparison to histology, the assay offered greater sensitivity and accuracy with 10/19 (53%) T. saginata positives detected by real-time PCR and none by histology. When the results were compared with the reference PCR, the assay was less sensitive but offered advantages of faster turnaround times and reduced contamination risk. Estimates of the assay's repeatability and reproducibility showed the assay is highly reliable with reliability coefficients greater than 0.94. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.

Genetic diversity and microcystin production by Anabaena in the Gulf of Finland, Baltic Sea

Syanobakteerit (sinilevät) ovat olleet Itämeressä koko nykymuotoisen Itämeren ajan, sillä paleolimnologiset todisteet niiden olemassaolosta Itämeren alueella ovat noin 7000 vuoden takaa. Syanobakteerien massaesiintymät eli kukinnat ovat kuitenkin sekä levinneet laajemmille alueille että tulleet voimakkaimmiksi viimeisten vuosikymmenien aikana. Tähän on osasyynä ihmisten aiheuttama kuormitus, joka rehevöittää Itämerta. Suomenlahti, jota tämä tutkimus käsittelee, on kärsinyt tästä rehevöitymiskehityksestä muita Itämeren altaita enemmän. Syanobakteerit muodostavat jokakesäisiä kukintoja Suomenlahdella - niin sen avomerialueilla kuin rannoillakin. Yleisimmät kukintoja muodostavat syanobakteerisuvut ovat Nodularia, Anabaena ja Aphanizomenon. Kukinnat aiheuttavat paitsi esteettistä haittaa myös terveydellisen riskitekijän. Niiden myrkyllisyys liitetään usein Nodularia-suvun tuottamaan nodulariini-maksamyrkkyyn. Itämeren Aphanizomenon-suvun on todettu olevan myrkytön. Vaikka Itämeren kukintoja aiheuttavista Nodularia- ja Aphanizomenon-syanobakteereista tiedetään varsin paljon, on molekyylimenetelmiin pohjautuva syanobakteeritutkimus ohittanut Itämeren Anabaena-suvun monelta osin. Tämän työn tarkoituksena oli syventää käsitystämme Itämeren Anabaena-syanobakteerista, sen mahdollisesta myrkyllisyydestä, geneettisestä monimuotoisuudesta ja fylogeneettisista sukulaisuussuhteista. Tässä työssä eristettiin 49 planktista Anabaena-kantaa, joista viisi tuottivat mikrokystiinejä. Tämä oli ensimmäinen yksiselitteinen todiste, että Itämeren Anabaena tuottaa maksamyrkyllisiä mikrokystiini-yhdisteitä. Jokainen eristetty myrkyllinen Anabaena-kanta tuotti useita mikrokystiini-variantteja. Lisäksi mikrokystiinejä löydettiin kukintanäytteistä, joissa oli myrkkyä syntetisoivia geenejä sisältäneitä Anabaena-syanobakteereita. Myrkkyjä löydettiin molempina tutkimusvuosina 2003 ja 2004. Myrkkyjen esiintyminen ei siten ollut vain yksittäinen ilmiö. Tässä työssä saimme viitteitä siitä, että maksamyrkyllinen Anabaena-syanobakteeri esiintyisi vähäsuolaisissa vesissä. Tämä riippuvuussuhde jää kuitenkin tulevien tutkimuksien selvitettäväksi. Tässä työssä havaittiin mikrokystiinisyntetaasi-geenien inaktivoituminen Itämeren Anabaena-kannassa ja kukintanäytteissä. Kuvasimme Anabaena-kannan mikrokystiinisyntetaasigeenien sisältä insertioita, jotka hyvin todennäköisesti inaktivoivat myrkyntuoton. Insertion sisältäneeltä kannalta löysimme kuitenkin kaikki mikrokystiinisyntetaasigeenit osoittaen, että geenien olemassaolo ei välttämättä varmista kannan mikrokystiinintuottoa. Mielenkiintoista oli se, että inaktivaation aiheuttavia insertioita löytyi kukintanäytteistä molemmilta tutkimusvuosilta. Vastaavia insertioita ei kuitenkaan löydetty makean veden Anabaena-kannoista tai järvinäytteistä. On yleistä, että syanobakteerikukinnoista löytyy usean syanobakteerisuvun edustajia. Myrkyllisiä sukuja tai lajeja ei voida kuitenkaan erottaa mikroskooppisesti myrkyttömistä. Käsillä olevassa tutkimuksessa kehitettiin molekyylimenetelmä, jolla on mahdollista määrittää kukinnan mahdollisesti maksamyrkylliset syanobakteerisuvut. Tätä menetelmää sovellettiin Itämeren kukintojen tutkimiseen. Itämeren pintavesistä ja ranta-alueiden pohjasta eristetyt Anabaena-kannat osoittautuivat geneettisesti monimuotoisiksi. Tämä Anabaena-syanobakteerien geneettinen monimuotoisuus vahvistettiin monistamalla geenejä suoraan kukintanäytteistä ilman kantojen eristystä. Makeiden vesien ja Itämeren Anabaena-kannat ovat geneettisesti hyvin samankaltaisia. Geneettisissä vertailuissa kävi kuitenkin ilmi, että pohjassa elävien Anabaena-kantojen geneettinen monimuotoisuus oli suurempaa kuin pintavesistä eristettyjen kantojen. Itämeren Anabaena-kantojen sekvenssit muodostivat omia ryhmiä sukupuun sisällä, jolloin on mahdollista, että nämä edustavat Itämeren omia Anabaena-ekotyyppejä. Tämä tutkimus oli ensimmäinen, jossa uusin molekyylimenetelmin systemaattisesti selvitettiin Itämeren Anabaena-syanobakteerin geneettistä populaatiorakennetta, fylogeniaa ja myrkyntuottoa. Tulevaisuudessa monitorointitutkimuksissa on otettava huomioon myös Itämeren Anabaena-syanobakteerin mahdollinen maksamyrkyntuotto – erityisesti vähäsuolaisemmilla rannikkovesillä.

Biodiversity and Phylogeny of Planktic Cyanobacteria in Temperate Freshwater Lakes

Currently, the classification used for cyanobacteria is based mainly on morphology. In many cases the classification is known to be incongruent with the phylogeny of cyanobacteria. The evaluation of this classification is complicated by the fact that numerous strains are only described morphologically and have not been isolated. Moreover, the phenotype of many cyanobacterial strains alters during prolonged laboratory cultivation. In this thesis, cyanobacterial strains were isolated from lakes (mainly Lake Tuusulanjärvi) and both morphology and phylogeny of the isolates were investigated. The cyanobacterial community composition in Lake Tuusulanjärvi was followed for two years in order to relate the success of cyanobacterial phenotypes and genotypes to environmental conditions. In addition, molecular biological methods were compared with traditional microscopic enumeration and their ability and usefulness in describing the cyanobacterial diversity was evaluated. The Anabaena, Aphanizomenon, and Trichormus strains were genetically heterogeneous and polyphyletic. The phylogenetic relationships of the heterocytous cyanobacteria were not congruent with their classification. In contrast to heterocytous cyanobacteria, the phylogenetic relationships of the Snowella and Woronichinia strains, which had not been studied before this thesis, reflected the morphology of strains and followed their current classification. The Snowella strains formed a monophyletic cluster, which was most closely related to the Woronichinia strain. In addition, a new cluster of thin, filamentous cyanobacterial strains identified as Limnothrix redekei was revealed. This cluster was not closely related to any other known cyanobacteria. The cyanobacterial community composition in Lake Tuusulanjärvi was studied with molecular methods [denaturant gradient gel electrophoresis (DGGE) and cloning of the 16S rRNA gene], through enumerations of cyanobacteria under microscope, and by strain isolations. Microcystis, Anabaena/Aphanizomenon, and Synechococcus were the major groups in the cyanobacterial community in Lake Tuusulanjärvi during the two-year monitoring period. These groups showed seasonal succession, and their success was related to different environmental conditions. The major groups of the cyanobacterial community were detected by all used methods. However, cloning gave higher estimates than microscopy for the proportions of heterocytous cyanobacteria and Synechococcus. The differences were probably caused by the high 16S rRNA gene copy numbers in heterotrophic cyanobacteria and by problems in the identification and detection of unicellular cyanobacteria.

Status, challenges and tools for identification and diagnosis of Peronosclerospora and Sclerophthora of regulatory concern for graminicolous crops

Graminicolous Downy Mildew (GDM) diseases caused by the genera Peronosclerospora (13 spp.) and Sclerophthora (6 spp. and 1 variety) are poorly studied but destructive diseases of major crops such as corn, sorghum, sugarcane and other graminoids. Eight of the 13 described Peronosclerospora spp. are able to infect corn. In particular, P. philippinensis (= P. sacchari), P. maydis, P. heteropogonis, and S. rayssiae var. zeae cause major losses in corn yields in tropical Asia. In 2012 a new species, P. australiensis, was described based on isolates previously identified as P. maydis in Australia; this species is now a pathogen of major concern. Despite the strong impact of GDM diseases, there are presently no reliable molecular methods available for their detection. GDM pathogens are among the most difficult Oomycetes to identify using molecular tools, as their taxonomy is very challenging, and little genetic sequence data are available for development of molecular tools to detect GDM pathogens to species level. For example, from over 15 genes used in identification, diagnostics or phylogeny of Phytophthora, only ITS1 and cox2 show promise for use with GDM pathogens. Multiplex/multigene conventional and qPCR assays are currently under evaluation for the detection of economically important GDM spp. Scientists from the USA, Germany, Canada, Australia, and the Philippines are collaborating on the development and testing of diagnostic tools for these pathogens of concern.

Phylogenetics of Myrmica ants and their social parasites

Understanding the overwhelming diversity of life calls for complex organisational schemes. The field of systematics may thus be seen as the cornerstone of evolutionary biology. In the last few decades, systematics has been rejuvenated through the introduction of molecular methods such as DNA barcoding and multi-gene phylogenetic approaches. These methods may shed new light on established taxonomic ideas and problems. For example, the classification of ants has aroused much debate due to reinterpretation of morphological characters or contradictions between molecular data and morphology. Only in the last few years a consensus was reached regarding the phylogeny of ant subfamilies. However, the situation remains deplorable for lower taxonomic ranks such as subfamilies, tribes and genera. This thesis describes the systematics and evolution of the Holarctic ant genus Myrmica and the tribe to which it belongs, Myrmicini. Using barcoding, molecular-phylogenetic data and divergence time estimations, it addresses questions regarding the taxonomy, morphology and biogeography of this group. Furthermore, the interrelationships between socially parasitic Myrmica species and their hosts (other species in the genus) were inferred. The phylogeny suggests that social parasitism evolved several times in Myrmica. Finally, this thesis investigated whether coevolution shaped the phylogeny of socially parasitic Maculinea butterflies that live inside Myrmica colonies. No evidence was found for coevolution.

Epidemiology of candidemia in Finland

Candida species are an important cause of nosocomial bloodstream infections in hospitalized patients worldwide, with associated high mortality, excess length of stay and costs. Main contributors to candidemias is profound immunosuppression due to serious underlying condition or intensive treatments leading to an increasing number of susceptible patients. The rank order of causative Candida species varies over time and in different geographic locations. The aim of this study was to obtain information on epidemiology of candidemia in Finland, to identify trends in incidence, causative species, and patient populations at risk. In order to reveal possible outbreaks and assess the value of one molecular typing method, restriction enzyme analysis (REA), in epidemiological study, we analyzed C. albicans bloodstream isolates in Uusimaa region in Southern Finland during eight years. The data from the National Infectious Disease Register were used to assess the incidence and epidemiological features of candidemia cases. In Helsinki University Central Hospital (HUCH) all patients with blood culture yielding any Candida spp. were identified from laboratory log-books and from Finnish Hospital Infection Program. All the patients with a stored blood culture isolate of C. albicans were identified through microbiology laboratory logbooks, and stored isolates were genotyped with REA in the National Institute for Health and Welfare (former KTL). The incidence of candidemia in Finland is globally relatively low, but increased between between 1990s and 2000s. The incidence was highest in males >65 years of age, but incidence rates for patients <1-15 years were lower during 2000s than during 1990s. In HUCH the incidence of candidemia remained low and constant during our 18 years of observation, but a significant shift in patient-populations at risk was observed, associated with patients treated in intensive care units, such as premature neonates and surgical patients. The predominating causative species in Finland and in HUCH is C. albicans, but the proportion of C. glabrata increased considerably. The crude one-month case fatality was constantly high between 28-33%. REA differentiated efficiently between C. albicans blood culture isolates and no clusters were observed in the hospitals involved, despite of abundant transfer of patients among them. Candida spp. are an important cause of nosocomial blood stream infections in Finland, and continued surveillance is necessary to determine the overall trends and patient groups at risk, and reduce the impact of these infections in the future. Molecular methods provide an efficient tool for investigation of suspected outbreak and should be available in the future in Finland, also.

Microbial identification by detection of ligation probes on DNA microarray

Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection.