A novel TaqMan qPCR assay for rapid detection and quantification of pro-inflammatory microalgae Prototheca spp. in milk samples.

Animal or human protothecosis belongs to rather rare, endemic, pro-inflammatory infections. It is caused by achlorophyllous algae of the genus Prototheca. Especially, P. bovis (formerly P. zopfii genotype 2) is often inflected as a non-bacterial causative agent of dairy cattle mastitis. In this study, we present a multiplex real-time PCR (qPCR) system for rapid and exact Prototheca spp. detection and quantification. Limit of detection, diagnostic sensitivity, and specificity were determined. For the first time, specific sequences of AccD (encoding acetyl CoA reductase) for P. bovis, cox1 (encoding cytochrome C oxidase subunit 1) for P. wickerhamii, cytB (encoding cytochrome B) for P. blashkeae and atp6 (encoding transporting ATPase F0 subunit 6) for P. ciferrii (formerly P. zopfii genotype 1) were used for species identification and quantification together with 28S rRNA sequence detecting genus Prototheca. The developed qPCR assay was applied to 55 individual cow milk samples from a herd suspected of protothecosis, 41 bulk milk samples from different Czech farms, 16 boxed milk samples purchased in supermarkets and 21 environmental samples originating from a farm suspected of protothecosis. Our work thus offers the possibility to diagnose protothecosis in the samples, where bacterial mastitis is the most commonly presumed and thereby assisting adequate corrective measures to be taken.

Control of paratuberculosis: who, why and how. A review of 48 countries.

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.

The water environment as a source of potentially pathogenic mycobacteria.

Nontuberculous mycobacteria (NTM) are ubiquitous organisms of a wide variety of environmental reservoirs, including natural and municipal water, soil, aerosols, protozoans, animals and humans. Several of these species are potential pathogens which affect human health. The aim of this study was to determine the occurrence of NTM in the water environment. Samples were taken from 13 water-related facilities including fish ponds, storage ponds, drinking water reservoirs and an experimental recirculation system. Altogether, 396 samples of water, sediment and aquatic plants were collected and analysed. All samples were examined using conventional culture methods. Suspected microbial isolates were subjected to polymerase chain reaction analysis and identified using partial sequence analysis of the 16S rDNA gene. The culture revealed 94/396 samples (23.7%) that contained mycobacteria. Among known NTM we identified potentially pathogenic mycobacteria isolated from the fresh water environment for the first time: Mycobacterium asiaticum, M. chimaera, M. interjectum, M. kumamotonense, M. lentiflavum, M. montefiorense, M. nebraskense, M. paraffinicum and M. simiae. Epidemiologic studies suggest that the natural water environment is the principal source of human exposure. Our results indicate that besides the well-known potentially pathogenic mycobacteria it is important to observe occurrence, proliferation and persistence of newly discovered mycobacterial species.

Substantial decrease in SARS-CoV-2 RNA after fixation of cadavers intended for anatomical dissection.

With the onset of the COVID-19 pandemic, a problem arose with classic body donation programmes for obtaining cadavers for anatomical dissections, science and research. The question has emerged whether bodies of individuals who died of COVID-19 or were infected by SARS-CoV-2 could be admitted to Departments of Anatomy. To determine the risk of SARS-CoV-2 transmission to employees or students, the presence and stability of SARS-CoV-2 RNA in cadavers after fixation agents' application and subsequent post-fixation baths over time were examined. The presence of viral RNA in swabs from selected tissues was assessed by the standardized routine RNA isolation protocol and subsequent real-time PCR analysis. To support the results obtained from the tissue swabs, samples of RNA were exposed in vitro to short and long-term exposure to the components of the injection and fixation solutions used for the bodies' conservation. Substantial removal of SARS-CoV-2 RNA was observed in post-mortem tissue following perfusion with 3.5% phenol, 2.2% formaldehyde, 11.8% glycerol and 55% ethanol, and subsequent post-fixation in an ethanol bath. In vitro experiments showed significant effects of formaldehyde on SARS-CoV-2 RNA, while phenol and ethanol showed only negligible effects. We conclude that cadavers subjected to fixation protocols as described here should not pose a considerable risk of SARS-CoV-2 infection while being handled by students and staff and are, therefore, suitable for routine anatomical dissections and teaching.

Hepatitis E virus in pork production chain in Czech Republic, Italy, and Spain, 2010.

We evaluated the prevalence of hepatitis E virus (HEV) in the pork production chain in Czech Republic, Italy, and Spain during 2010. A total of 337 fecal, liver, and meat samples from animals at slaughterhouses were tested for HEV by real-time quantitative PCR. Overall, HEV was higher in Italy (53%) and Spain (39%) than in Czech Republic (7.5%). HEV was detected most frequently in feces in Italy (41%) and Spain (39%) and in liver (5%) and meat (2.5%) in Czech Republic. Of 313 sausages sampled at processing and point of sale, HEV was detected only in Spain (6%). HEV sequencing confirmed only g3 HEV strains. Indicator virus (porcine adenovirus) was ubiquitous in fecal samples and absent in liver samples and was detected in 1 slaughterhouse meat sample. At point of sale, we found porcine adenovirus in sausages (1%-2%). The possible dissemination of HEV and other fecal viruses through pork production demands containment measures.

A viability assay combining palladium compound treatment with quantitative PCR to detect viable Mycobacterium avium subsp. paratuberculosis cells.

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.

Identification of and discrimination between the Mycobacterium abscessus complex and Mycobacterium avium complex directly from sputum using quadruplex real-time PCR.

Introduction. Cystic fibrosis (CF) is a serious disease with multisystemic clinical signs that is easily and frequently complicated by bacterial infection. Recently, the prevalence of nontuberculous mycobacteria as secondary contaminants of CF has increased, with the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics.Gap statement. Sensitive method for early detection and differentiation of MABSC members and MAC complex for use in routine clinical laboratories is lacking. A method based on direct DNA isolation from sputum, using standard equipment in clinical laboratories and allowing uncovering of possible sample inhibition (false negative results) would be required. The availability of such a method would allow accurate and accelerated time detection of MABSC members and their timely and targeted treatment.Aim. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex.Methodology. The method of DNA isolation directly from the sputum of patients followed by quadruplex real-time quantitative PCR (qPCR) detection was developed and optimised. The sensitivity and limit of detection (LOD) of the qPCR was determined using human sputum samples artificially spiked with a known amount of M. abscessus subsp. massiliense (MAM).Results. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between M. abscessus subsp. abscessus/subsp. bolletii (MAAb/MAB) and MAM. The system was verified using 61 culture isolates and sputum samples from CF and non-CF patients showing 29.5 % MAAb/MAB, 14.7 % MAM and 26.2 % MAC. The LOD was determined to be 1 490 MAM cells in the sputum sample with the efficiency of DNA isolation being 95.4 %. Verification of the qPCR results with sequencing showed 100 % homology.Conclusions. The developed quadruplex qPCR assay, which is preceded by DNA extraction directly from patients' sputum without the need for culturing, significantly improves and speeds up the entire process of diagnosing CF patients and is therefore particularly suitable for use in routine laboratories.

Six genetically linked mutations in the CD36 gene significantly delay the onset of Alzheimer's disease.

The risk of Alzheimer's disease (AD) has a strong genetic component, also in the case of late-onset AD (LOAD). Attempts to sequence whole genome in large populations of subjects have identified only a few mutations common to most of the patients with AD. Targeting smaller well-characterized groups of subjects where specific genetic variations in selected genes could be related to precisely defined psychological traits typical of dementia is needed to better understand the heritability of AD. More than one thousand participants, categorized according to cognitive deficits, were assessed using 14 psychometric tests evaluating performance in five cognitive domains (attention/working memory, memory, language, executive functions, visuospatial functions). CD36 was selected as a gene previously shown to be implicated in the etiology of AD. A total of 174 polymorphisms were tested for associations with cognition-related traits and other AD-relevant data using the next generation sequencing. Several associations between single nucleotide polymorphisms (SNP's) and the cognitive deficits have been found (rs12667404 with language performance, rs3211827 and rs41272372 with executive functions, rs137984792 with visuospatial performance). The most prominent association was found between a group of genotypes in six genetically linked and the age at which the AD patients presented with, or developed, a full-blown dementia. The identified alleles appear to be associated with a delay in the onset of LOAD. In silico studies suggested that the SNP's alter the expression of CD36 thus potentially affecting CD36-related neuroinflammation and other molecular and cellular mechanisms known to be involved in the neuronal loss leading to AD. The main outcome of the study is an identification of a set of six new mutations apparently conferring a distinct protection against AD and delaying the onset by about 8 years. Additional mutations in CD36 associated with certain traits characteristic of the cognitive decline in AD have also been found.

"Mycobacterium avium subsp. hominissuis" in neck lymph nodes of children and their environment examined by culture and triplex quantitative real-time PCR.

"Mycobacterium avium subsp. hominissuis" often causes cervical lymphadenitis in children; its prompt and accurate identification enables adequate therapy, tracing, and prevention. The aims of this study were to determine the causative agent of lymphadenitis using culture, PCR, and triplex quantitative real-time PCR (qPCR) methods with DNA directly isolated from tissue, as well as to identify possible sources of infection from the environment. We confirmed the diagnoses by detecting M. avium subsp. hominissuis using qPCR with DNA directly isolated from lymph node biopsy specimens of two patients. In order to trace the source of infection from the environment, a method of DNA isolation from soil and other environmental samples, such as dust, cobwebs, and compost, was developed. The triplex qPCR examination revealed the presence of M. avium subsp. hominissuis in a high proportion of the environmental samples (42.8% in the first patient's house and 47.6% in the second patient's house). Both patients were also exposed to M. avium subsp. avium, which was present due to the breeding of infected domestic hens. The high infectious dose of M. avium subsp. hominissuis or the increased susceptibility of humans to M. avium subsp. hominissuis compared to M. avium subsp. avium could be the reason why the children were infected with M. avium subsp. hominissuis.

Outbreak of Mycobacterium avium subsp. avium infection in one flock of domestic pigeons.

An outbreak of Mycobacterium avium subsp. avium infection was diagnosed in one breed of domestic pigeons (Columba livia f. domestica) in the Czech Republic. Nodular granulomatous lesions were found in 42 (9.7%) pigeons of the 435 examined; histopathologic examination of livers with gross lesions of mycobacteriosis from 15 randomly selected pigeons revealed granulomatous inflammation typical for avian mycobacteriosis in all samples. Direct Ziehl-Neelsen (ZN) microscopy and conventional culture were performed for a total of 117 liver samples (42 pigeons with nodular lesions, 55 randomly selected pigeons without nodular lesions, and 20 randomly selected squabs). Acid-fast bacilli were observed in 19 (16.2%), and conventional culture yielded growth of M. a. avium in 40 (34.2%) liver samples. A triplex quantitative real-time PCR assay based on the IS901 detection system was performed successfully in 115 liver samples and revealed M. a. avium in 63 (54.8%) of them. Mycobacterium a. avium was also detected in two squabs. Eight domestic rabbits (Oryctolagus cuniculus f. domestica) living in the breeding facility were also examined. Pyogranulomatous lesions were only found in one adult male rabbit. At necropsy, both direct ZN microscopy and culture gave negative results for mycobacteria in all examined rabbit tissues. Mycobacterium a. avium was diagnosed in a liver sample of one juvenile rabbit using triplex qPCR, suggesting that M. a. avium infection can occur as early as juvenile animals.

Mycobacterium avium subsp. Paratuberculosis and the expression of selected virulence and pathogenesis genes in response to 6°C, 65°c and ph 2.0.

The aim of this work was to study the expression of selected Mycobacterium avium subsp. paratuberculosis (MAP) genes connected with MAP virulence, adhesion and stress response. The temperature of 6°C and 65°C were chosen with regard to the food industry, storage conditions (refrigerator) and low-temperature pasteurization. A pH of 2.0, using lactic acid, was selected to mimic the natural environment of the stomach. Expression of selected genes was studied using real time reverse transcription PCR on three different MAP isolates. MAP isolates were chosen according to the number of their preceding cultivations. While isolates 8672 and 8819 were previously cultivated only once, MAP isolate 12146 went through four passages. Different expression profiles were observed in each of the three MAP isolates. However, particular similar patterns were observed. SigE, sigF and ahpC were up-regulated, while sigL was down-regulated under temperature stress. Mmp gene was found to be down-regulated under acidic conditions. Low passage isolates (8672 and 8819) showed certain level of acid resistance.

Occurrence of SARS-CoV-2 in Indoor Environments With Increased Circulation and Gathering of People.

At the time of sampling (2020/2021), the number of new cases of SARS-CoV-2-positive individuals in the Czech Republic significantly exceeded the numbers in neighboring countries and in the EU. In terms of the number of deaths, the country ranked near the top of the list. Legislative orders required wearing masks indoors, disinfecting surfaces in public places, and limiting the number of people per sales area in commercial spaces. Due to an situation, most schools and shops were closed. The entire country anticipated a total lockdown. To assess the risk to public health regarding SARS-CoV-2 transmission, air and surfaces were sampled in two public places: a post office and a shopping center. Samples were also collected at the COVID-19 unit at the local hospital. Neither air nor surface samples were positive for SARS-CoV-2 virus particles in the post office or shopping center. Positive results were found in the hospital ward, with floors being the most and highest contaminated surface. Based on our results, we believe that public places do not pose a risk in relation to SARS-CoV-2 transmission, especially when epidemiological measures to reduce transmission are followed, such as wearing masks, using disinfectant or limiting the number of customers per retail establishment.

A Novel Approach to the Viability Determination of Mycobacterium avium subsp. paratuberculosis Using Platinum Compounds in Combination With Quantitative PCR.

Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 µM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.

MOL-PCR and xMAP Technology: A Multiplex System for Fast Detection of Food- and Waterborne Viruses.

Viruses are common causes of food- and waterborne diseases worldwide. Conventional identification of these agents is based on cultivation, antigen detection, electron microscopy, or real-time PCR. Because recent technological advancements in detection methods are focused on fast and robust analysis, a rapid multiplexing technology, which can detect a broad spectrum of pathogenic viruses connected to food or water contamination, was utilized. A new semiquantitative magnetic bead-based multiplex system has been designed for simultaneous detection of several targets in one reaction. The system includes adenoviruses 40/41 (AdV), rotavirus A (RVA), norovirus (NoV), hepatitis E virus (HEV), hepatitis A virus (HAV), and a target for external control of the system. To evaluate the detection system, interlaboratory ring tests were performed in four independent laboratories. Analytical specificity of the tool was tested on a cohort of pathogenic agents and biological samples with quantitative PCR as a reference method. Limit of detection (analytical sensitivity) of 5 × 100 (AdV, HEV, and RVA) and 5 × 101 (HAV and NoV) genome equivalents per reaction was reached. This robust, senstivie, and rapid multiplexing technology may be used to routinely monitor and manage viruses in food and water to prevent food and waterborne diseases.

Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR.

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

Consecutive excretion of Mycobacterium avium subspecies paratuberculosis in semen of a breeding bull compared to the distribution in feces, tissue and blood by IS900 and F57 quantitative real-time PCR and culture examinations.

Paratuberculosis (Johne's disease) has emerged as one of the most important diseases in cattle. The role of infected bull semen in the spread of infection remains unclear, as the correlation between the amount of excreted Mycobacterium avium subsp. paratuberculosis (semen and feces) and the infection load (blood and tissues) has not been defined. The aim of the present study was to study by culture, and a quantitative real-time polymerase chain reaction, the presence of bacteria in consecutive semen, blood, and fecal samples collected from one infected Piedmont breeding bull during a 380-day period. Five out of seven blood samples and all nine semen samples were positive in the real-time quantitative polymerase chain reaction with 10¹ to 10² and 10² to 104 copies of IS900/F57 per ml, respectively. In all, there were 9 fecal culture positive samples with too numerous to count colony forming units and positive real-time quantitative polymerase chain reactions ranging from 105 to 107 copies of IS900/F57. After the bull was euthanized, Mycobacterium avium subsp. paratuberculosis was cultured from various parts of the small and large intestines, liver tissue and lymph nodes and from the epididymis and vesicular glands. The results demonstrate a wide extraintestinal distribution of the bacterium and that breeding bulls should be considered a source of paratuberculosis infection due to their contact with other breeding bulls and a high number of heifers and cows through the natural mating process.

Efficiency of DNA Isolation Methods Based on Silica Columns and Magnetic Separation Tested for the Detection of Mycobacterium avium Subsp. Paratuberculosis in Milk and Faeces.

Timely and reliable detection of animals shedding Mycobacterium avium subsp. paratuberculosis (MAP) should help to effectively identify infected animals and limit infection transmission at early stages to ensure effective control of paratuberculosis. The aim of the study was to compare DNA extraction methods and evaluate isolation efficiency using milk and faecal samples artificially contaminated by MAP with a focus on modern instrumental automatic DNA isolation procedures based on magnetic separation. In parallel, an automatic and manual version of magnetic separation and two methods of faecal samples preparation were compared. Commercially available DNA isolation kits were evaluated, and the selected kits were used in a trial of automatic magnetic beads-based isolation and compared with the manual version of each kit. Detection of the single copy element F57 was performed by qPCR to quantify MAP and determine the isolation efficiency. The evaluated kits showed significant differences in DNA isolation efficiencies. The best results were observed with the silica column Blood and Tissue kit for milk and Zymo Research for faeces. The highest isolation efficiency for magnetic separation was achieved with MagMAX for both matrices. The magnetic separation and silica column isolation methods used in this study represent frequently used methods in mycobacterial diagnostics.

Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR.

Quantitative PCR (qPCR) is a widely used method for nucleic acid quantification of various pathogenic microorganisms. For absolute quantification of microbial load by qPCR, it is essential to create a calibration curve from accurately quantified quantification standards, from which the number of pathogens in a sample is derived. Spectrophotometric measurement of absorbance is a routine method for estimating nucleic acid concentration, however, it may be affected by presence of other potentially contaminating nucleic acids or proteins and salts. Therefore, absorbance measurement is not reliable for estimating the concentration of stock solutions of quantification standards, based on which they are subsequently diluted. In this study, we utilized digital PCR (dPCR) for absolute quantification of qPCR plasmid standards and thus detecting possible discrepancies in the determination of the plasmid DNA number of standards derived from UV spectrophotometry. The concept of dPCR utilization for quantification of standards was applied on 45 qPCR assays using droplet-based and chip-based dPCR platforms. Using dPCR, we found that spectrophotometry overestimated the concentrations of standard stock solutions in the majority of cases. Furthermore, batch-to-batch variation in standard quantity was revealed, as well as quantitative changes in standards over time. Finally, it was demonstrated that droplet-based dPCR is a suitable tool for achieving defined quantity of quantification plasmid standards and ensuring the quantity over time, which is crucial for acquiring homogenous, reproducible and comparable quantitative data by qPCR.

Development and Inter-Laboratory Validation of Diagnostics Panel for Detection of Biothreat Bacteria Based on MOL-PCR Assay.

Early detection of biohazardous bacteria that can be misused as biological weapons is one of the most important measures to prevent the spread and outbreak of biological warfare. For this reason, many instrument platforms need to be introduced into operation in the field of biological warfare detection. Therefore the purpose of this study is to establish a new detection panel for biothreat bacteria (Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and confirm it by collaborative validation by using a multiplex oligonucleotide ligation followed by polymerase chain reaction and hybridization to microspheres by MagPix detection platform (MOL-PCR). Appropriate specific sequences in bacterial DNA were selected and tested to assemble the detection panel, and MOLigo probes (short specific oligonucleotides) were designed to show no cross-reactivity when tested between bacteria and to decrease the background signal measurement on the MagPix platform. During testing, sensitivity was assessed for all target bacteria using serially diluted DNA and was determined to be at least 0.5 ng/µL. For use as a diagnostic kit and easier handling, the storage stability of ligation premixes (MOLigo probe mixes) was tested. This highly multiplex method can be used for rapid screening to prevent outbreaks arising from the use of bacterial strains for bioterrorism, because time of analysis take under 4 h.

Oligonucleotide microarray technology and its application to Mycobacterium avium subsp. paratuberculosis research: a review.

Microarrays represent a modern powerful technology, which have potential applications in many areas of biological research and provide new insights into the genomics and transcriptomics of living systems. The aim of this review is to describe the application of microarray technology for Mycobacterium avium subsp. paratuberculosis (MAP) research. The main focus points include a summary of results obtained for MAP using microarrays, examination of the fields of MAP research which are currently being investigated and possible areas of future research. This article is divided into two parts according to the type of nucleic acid used for array hybridisation. Articles related to MAP research using microarray technology are then divided according to the field of study, such as comparative genome analysis, diagnostics, expression or environmental studies.