TaqMan qPCR Assay Research Articles

Application of TaqMan Real-Time PCR for Detecting 'Candidatus Arsenophonus Phytopathogenicus' Infection in Sugar Beet.

The γ-proteobacterium ‘Candidatus Arsenophonus phytopathogenicus’ is assigned as the major pathogen of “Syndrome des basses richesses”, a sugar beet disease characterised by a reduction in the sugar content of taproots and biomass yield. Despite the economic impact of this bacteriosis, diagnostics for this important pathogen currently rely on end-point PCR detection. Herein, we introduce a TaqMan qPCR for diagnostics of the agent targeting genes encoding a heat shock protein of the Hsp20 family and mannose-6-phosphate isomerase. Quantitation with synthetic oligonucleotides as standard showed that the developed TaqMan qPCR assays enable the detection of up to 100 target copies. A comparison between the TaqMan qPCR and end-point PCR for ‘Ca. A. phytopathogenicus’ detection was carried out on 78 sugar beet samples from different locations in southern Germany. The newly developed assays enable the fast, reliable and sensitive detection of ‘Ca. A. phytopathogenicus’ in sugar beet.

Development and Validation of Two Diagnostic Real-Time PCR (TaqMan) Assays for the Detection of Bordetella avium from Clinical Samples and Comparison to the Currently Available Real-Time TaqMan PCR Assay

Bordetella avium (BA) is one of many pathogens that cause respiratory diseases in turkeys. However, other bacterial species can easily overgrow it during isolation attempts. This makes confirming the diagnosis of BA as the causative agent of turkey coryza more difficult. Currently, there are two PCR assays for the molecular detection of BA. One is conventional gel-based PCR and the other is TaqMan real-time PCR (qPCR) assay. However, multiple pitfalls were detected in both assays regarding their specificity, sensitivity, and efficiency, which limits their utility as diagnostic tools. In this study, we developed and validated two TaqMan qPCR assays and compared their performance to the currently available TaqMan qPCR. The two assays were able to correctly identify all BA isolates and showed negative results against a wide range of different microorganisms. The two assays were found to have high efficiency with a detection limit of approximately 1 × 103 plasmid DNA Copies/mL with high repeatability and reproducibility. In comparison to the currently available TaqMan qPCR assay, the newly developed assays showed significantly higher PCR efficiencies due to superior primers and probes design. The new assays can serve as a reliable tool for the sensitive, specific, and efficient diagnosis of BA.

Multiple TaqMan qPCR and droplet digital PCR (ddPCR) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae.

Decisions on which pesticide to use in agriculture are expected to become more difficult, as the number of available chemicals is decreasing. For Tetranychus urticae (T. urticae), a major pest for which a number of candidate markers for pesticide resistance are in place, molecular diagnostics could support decision-making for the rational use of acaricides. A suite of 12 TaqMan qPCR assays [G314D (GluCl1), G326E, I321T (GluCl3), G119S, F331W (Ace-1), H92R (PSST), L1024V, F1538I (VGSC), I1017F (CHS1), G126S, S141F, P262T (cytb)], were validated against Sanger-sequencing, and subsequently adapted for use with the ddPCR technology. The concordance correlation coefficient between the actual and ddPCR measured mutant allelic frequencies was 0.995 (95% CI=0.991-0.998), and no systematic, proportional, or random differences were detected. The achieved Limit of Detection (LoD) was 0.1% (detection of one mutant in a background of 999 wild type mites). The ddPCR assay panel was then assessed in terms of agreement with phenotypic resistance, through a pilot application in field populations from Crete, with strong correlation and thus predictive and diagnostic value of the molecular assays in some cases (e.g., etoxazole and abamectin resistance). Molecular diagnostics were able to capture incipient resistance that was otherwise missed by phenotypic bioassays. The molecular and phenotypic resistance screening of T. urticae field populations from Crete, revealed both multi-resistant and susceptible populations. The highly sensitive T. urticae molecular diagnostic platforms developed in this study could prove a valuable tool for pesticide resistance management. © 2021 Society of Chemical Industry.

Two new superior primer pairs for universal detection of Xylella spp. in conventional PCR and TaqMan quantitative real-time PCR

Xylella fastidiosa causes many economically important plant diseases such as Pierce's disease of grapevine, citrus variegated chlorosis disease, and olive quick decline syndrome. Another species in the same genus, Xylella taiwanensis, causes pear leaf scorch. Here, to enable an initial screening of plants suspected of being infected with Xylella spp. by conventional polymerase chain reaction (cPCR), new primer pairs—X67S1/XL2r and XrDf1/XLr4—were designed to target the 16S ribosomal DNA (rDNA) of not only X. fastidosa but also X. taiwanensis. In cPCR to detect both species, X67S1/XL2r showed features superior to those of other primer pairs, such as fewer false negatives and false positives, whereas XrDf1/XLr4 seemed to be unsuitable because of abundant non-specific amplification. However, when XrDf1/XLr4 was combined with a probe in a TaqMan quantitative real-time PCR (qPCR), the assay detected no false positives and was more useful in the universal detection of Xylella spp. than TaqMan qPCR assays reported previously.

Using bacterial whole genome sequencing to identify toxin genes associated with disease outbreaks in black tiger shrimp (Penaeus monodon) aquaculture production

Vibrio-related diseases cause significant economic impact on global shrimp production. Although toxin expression is a well-established mechanism of Vibrio-related disease in humans, the role of toxin expression in Vibrio disease in aquaculture generally is not well defined. The current study investigated a sustained, rapid onset, mass mortality at a commercial tiger prawn, Penaeus monodon, hatchery. Affected shrimp were between mysis-2 and post-larvae-15. Disease outbreaks were prevented through prophylactic treatment with oxytetracycline, suggesting a bacterial-related causative agent. Conversely, mortality was not reduced with erythromycin treatment. Whole genome sequencing (WGS) using the Illumina Nextera XT protocol performed on mixed microbial cultures from erythromycin and non-erythromycin infused thiosulphate-citrate-bile salts-sucrose (TCBS) TCBS agar on a MiSeq, and postlarval samples on a HiSeq. The WGS revealed several Vibrio-related sequences dominated by reads with highest homology to Vibrio harveyi and Vibrio alginolyticus. In addition, a range of putative virulence genes encoding for antibiotic resistance and toxin production were identified. Taqman qPCR assays were designed to specifically quantify four of the putative toxin-genes found, namely the Zonula occludens toxin (ZOT), hemolysin d toxin (HylD), YaFO toxin-antitoxin and Repeats–in-Toxin (RTX) genes. The qPCR assays confirmed the presence of the toxin target genes with low cycle threshold value (Ct) in the bacterial cultures and postlarval samples. Specifically, the ZOT and HylD genes were detected in high copy number in the non-erythromycin and erythromycin-treated cultures. Although the detection of toxin-genes does not demonstrate causation of mortality, the application of WGS combined with qPCR analysis has revealed the presence of a range of virulence factors within the hatchery system and allowed a rapid implementation of management protocols to prevent entry of genetically toxin-armed Vibrio spp. until more conclusive studies into disease mechanisms could be conducted.

Development of a TaqMan qPCR assay for the detection and quantification of Gnomoniopsis castaneae in chestnut tissues

AbstractA novel real‐time PCR assay based on the TaqMan probe was developed for the detection of Gnomoniopsis castaneae, causal agent of brown rot of chestnut kernels, and responsible for leaf necrosis, shoot blight and bark canker. A part of the pathogen life cycle is endophytic, colonizing all tissues of chestnut and additional hosts, which is suspected to play a key role in its epidemiology. Thus, a molecular tool for sensitive detection and quantification of G. castaneae in symptomatic and asymptomatic host tissues is urgently required to better understand G. castaneae ecology, biology and epidemiology. Primers and a species‐specific probe for G. castaneae were designed based on the sequence of the single‐copy elongation factor 1 alpha (EF1α) gene. The amplification efficiency of target DNA was 105.3% and the limit of detection of the assay was calculated at approximately 40 fg of pure fungal DNA. The pathogen was consistently detected in artificial mixtures of plant and pathogen DNAs with the same Limit of Detection (LOD) as pure fungal DNA. In naturally infected samples, the assay rapidly revealed the presence of the pathogen in all symptomatic specimens, as well as in asymptomatic tissues. Notably, a significant relationship between the results of a metagenomic HTS analysis and the qPCR assay on DNAs extracted from bulk fruit was found. This molecular tool will be of substantial aid in detecting and quantifying G. castaneae, even in the endophytic state, and in different host tissues.

Dynamics of Specific cfDNA Fragments in the Plasma of Full Marathon Participants.

Plasma cell-free DNA (cfDNA) is frequently analyzed using liquid biopsy to investigate cancer markers. We hypothesized that this concept might be applicable in exercise physiology. Here, we aimed to identify specific cfDNA (spcfDNA) sequences in the plasma of healthy humans using next-generation sequencing (NGS) and clearly define the dynamics regarding spcfDNA-fragment levels upon extreme exercises, such as running a full marathon. NGS analysis was performed using cfDNA of pooled plasma collected from healthy participants. We confirmed that the TaqMan-qPCR assay had high sensitivity and found that the spcfDNA sequence abundance was 16,600-fold higher than that in a normal genomic region. We then used the TaqMan-qPCR assay to investigate the dynamics of spcfDNA-fragment levels upon running a full marathon. The spcfDNA fragment levels were significantly increased post-marathon. Furthermore, spcfDNA fragment levels were strongly correlated with white blood cell and plasma myoglobin concentrations. These results suggest the spcfDNA fragments identified in this study were highly sensitive as markers of extreme physical stress. The findings of this study may provide new insights into exercise physiology and genome biology in humans.

Multiple internal controls enhance reliability for PCR and real time PCR detection of Rathayibacter toxicus

Rathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa. A threat to livestock industries globally, the bacterium was designated a U.S. Select Agent. Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns. Whole genomes of three genetic populations of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus to the population level of discrimination. Specificity was confirmed with appropriate inclusivity and exclusivity panels; no cross reactivity was observed. The endpoint multiplex PCR and TaqMan qPCR assays detected 10 fg and 1 fg of genomic DNA, respectively. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls. Assays were validated by blind ring tests with multiple operators in three international laboratories.

Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells.

Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant's mode of action. To address the limited approaches available for identifying epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration-response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (RGL1, NEU1, GPR183) or cellular stress-response genes (ATF3, CDKN1A, GADD45A) analyzed by TaqMan qPCR assays after 4h of exposure. Whole transcriptomes were profiled after 4h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.

Tracing the almost extinct mayfly Prosopistoma pennigerum (Müller, 1785) – an eDNA approach

The rare mayfly Prosopistoma pennigerum was once widely distributed across Europe and occurred virtually in every large river. Today, it holds fast against the ever-growing destruction of its habitat with a few relic populations remaining. Preliminary data and information on its congeners suggest that free-flowing rivers with near-natural hydrodynamics are its primary habitat. Rivers where the species currently occurs should be primary targets for large-scale landscape conservation and protection. According to the Water Framework Directive, achieving longitudinal and lateral connectivity is a priority target, and occurrence and viability of Prosopistoma pennigerum populations could be an indicator of restoration success. We developed and validated a targeted qPCR protocol to detect this mayfly and applied it to standardized water samples filtered for eDNA analysis from the Vjosë (Albania) and Volga (Russia), two rivers with extant populations of P. pennigerum. In the Vjosë river, 45 sampling sites were sampled three times in 2018 and 2019. In the Volga river, we focused on a site with >15 years of continuous records of P. pennigerum at Rzhev and two downstream locations, where eDNA samples were collected in 2017. At each sampling site in the Vjosë, eDNA samples were collected by filtering 0.5 L of stream water through each of two 0.45 µm Sterivex filters. In the Volga, 2 L of stream water were filtered through a total of eight 0.7 µm glass fibre filters. Filters were stored and shipped at -20°C until further processing. Environmental DNA extraction was performed using the DNeasy® PowerWater® Sterivex™ Kit following the Experienced User protocol for Vjosë samples and via a Phenol-Chloroform Isoamyl extraction for the Volga samples. A Taqman qPCR assay was developed using a newly designed primer and probe set. Standard curves obtained from an amplicon dilution series yielded a reaction efficiency of 88% and an R2-value of 0.995, with a calculated limit of quantification of 1801 copies/µL and a limit of detection of 59 copies/µL. Each eDNA sampling replicate was tested using five qPCR replicates, yielding ten qPCR reactions per sampling site in the Vjosë river and 40 qPCR reactions in the Volga river. With each batch of eDNA samples, four negative and two positive controls were analysed. At each site, we also collected benthic MHS samples with a 25x25 cm 500 µm net where 20 samples were taken to reflect microhabitat distribution and dominance. Benthos samples were subsampled in the lab and all zoobenthos hand-picked. Prosopistoma pennigerum occurrence was assessed as areal density (number of specimens per m2). While P. pennigerum occured in high frequencies and abundances in benthic samples along the Vjosë river main stem in all sampling seasons, qPCR of eDNA samples suggested slightly different occurrence patterns. Conversely, P. pennigerum was detected with high consistency at two sites in the Volga river (at Rzhev and a site 99 km downstream), where its frequency is much lower than in the Vjosë river. The success of detecting a benthic species in eDNA samples depends on a variety of factors that may have affected DNA quality and prevented better detection of P. pennigerum in the Vjosë river eDNA samples. We demonstrate the principal applicability of molecular methods to search for rare species in hot-spots of biodiversity in Central Europe. The remnant populations of P. pennigerum in the hydrodynamically minimally impaired Vjosë and the Volga highlight the conservation and protection needs in Eastern and Southern Europe. At the European scale, restoration efforts should be geared towards creating viable habitat conditions for large-river species such as P. pennigerum. Here, our qPCR assay can deliver crucial data for better management of Europe’s large rivers.

A novel duplex qPCR assay for stepwise detection of multiple Perkinsea protistan infections of amphibian tissues.

Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.

Improved multiplex TaqMan qPCR assay with universal internal control offers reliable and accurate detection of Clavibacter michiganensis.

Clavibacter michiganensis (Cm) is a seed-borne plant pathogen that significantly reduces tomato production worldwide. Due to repeated outbreaks and rapid spread of the disease, seeds/transplants need to be certified free of the pathogen before planting. To this end, we developed a multiplex TaqMan qPCR assay that can accurately detect Cm in infected samples. A specific region of Cm (clvG gene) was selected for primer design using comparative genomics approach. A fully synthetic universal internal control (UIC) was also designed to detect PCR inhibitors and false-negative results in qPCRs. The Cm primers can be used alone or in a triplex TaqMan qPCR assay with UIC and previously described Clavibacter primers. The assay was specific for Cm and detected up to 10fg of Cm DNA in sensitivity and spiked assays. Addition of the UIC did not change the specificity or sensitivity of the multiplex TaqMan qPCR assay. The triplex TaqMan qPCR provides a specific and sensitive diagnostic assay for Cm. This assay can be used for biosecurity surveillance, routine diagnostics, estimating bacterial titres in infected material and for epidemiological studies. The UIC is fully synthetic, efficiently amplified and multiplex compatible with any other qPCR assay.

Systemic Helicobacter infection and associated mortalities in endangered Grand Cayman blue iguanas (Cyclura lewisi) and introduced green iguanas (Iguana iguana).

The Blue Iguana Recovery Programme maintains a captive breeding and head-starting program for endangered Grand Cayman blue iguanas (Cyclura lewisi) on Grand Cayman, Cayman Islands. In May 2015, program staff encountered two lethargic wild Grand Cayman blue iguanas within the Queen Elizabeth II Botanic Park (QEIIBP). Spiral-shaped bacteria were identified on peripheral blood smears from both animals, which molecular diagnostics identified as a novel Helicobacter species (provisionary name Helicobacter sp. GCBI1). Between March 2015 and February 2017, 11 Grand Cayman blue iguanas were identified with the infection. Two of these were found dead and nine were treated; five of the nine treated animals survived the initial infection. Phylogenetic analysis of the 16S rRNA gene suggests Helicobacter sp. GCBI1 is most closely related to Helicobacter spp. in chelonians. We developed a Taqman qPCR assay specific for Helicobacter sp. GCBI1 to screen tissue and/or blood samples from clinical cases, fecal and cloacal samples from clinically healthy Grand Cayman blue iguanas, including previously infected and recovered iguanas, and iguanas housed adjacent to clinical cases. Fecal and/or cloacal swab samples were all negative, suggesting that Grand Cayman blue iguanas do not asymptomatically carry this organism nor shed this pathogen per cloaca post infection. Retrospective analysis of a 2014 mortality event affecting green iguanas (Iguana iguana) from a separate Grand Cayman location identified Helicobacter sp. GCBI1 in two of three cases. The source of infection and mode of transmission are yet to be confirmed. Analysis of rainfall data reveal that all infections occurred during a multi-year dry period, and most occurred shortly after the first rains at the end of seasonal drought. Additionally, further screening has identified Helicobacter sp. GCBI1 from choanal swabs of clinically normal green iguanas in the QEIIBP, suggesting they could be asymptomatic carriers and a potential source of the pathogen.

Discriminating lineages of Batrachochytrium dendrobatidis using quantitative PCR.

The ability to detect and monitor infectious disease in a phylogenetically informative manner is critical for their management. Phylogenetically informative diagnostic tests enable patterns of pathogen introduction or changes in the distribution of genotypes to be measured, enabling research into the ecology of the pathogen. Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis in amphibian populations, emerged worldwide in the 21st century and is composed of six lineages which are display varying levels of virulence in their hosts. Research into the distribution, ecology and pathogenicity of these lineages has been hampered by an inability to type lineage efficiently. Here, we describe a lineage‐specific TaqMan qPCR assay that differentiates the two lineages of Bd most commonly associated with chytridiomycosis: BdGPL and BdCAPE. We demonstrate how this assay can be used for the surveillance of wild populations of amphibians in Southern Africa using skin swabs, tissue samples and cultured isolates.

Comparison of methods for the detection of Neisseria gonorrhoeae from South African women attending antenatal care.

The detection of Neisseria gonorrhoeae using culture assays is challenging. This study aims to compare different assays for the detection of N. gonorrhoeae. This cross-sectional study was conducted at King Edward VIII Hospital and included 307 antenatal attendees, each willing to provide two endocervical swabs. The first swab was used for culture identification of N. gonorrhoeae, and the second swab was processed for the detection of the pathogen by the TaqMan quantitative polymerase chain reaction (qPCR) assay, an in-house 16S ribosomal RNA (rRNA) PCR and PCR detection of the opa gene. Culture and the nucleic acid amplification assays were each used as comparator tests in the analysis. Sensitivity and specificity were calculated using RS Studio. The prevalence of N. gonorrhoeae was 7.8%. When compared to the TaqMan assay, the 16S rRNA PCR exhibited the highest sensitivity of 62%, with a substantial level of agreement (kappa level of agreement: 0.60), followed by the opa PCR (38%) with a moderate level of agreement (0.52) and culture exhibiting the lowest sensitivity of 25% with a fair level of agreement (0.38). The diagnostic accuracy of all the assays was >90%. The TaqMan qPCR assay has the ability to serve as a future diagnostic assay for the detection of N. gonorrhoeae.

Genetic Variation in the Bitter Receptors Responsible for Epicatechin Detection Are Associated with BMI in an Elderly Cohort.

Globally, more than one-third of adults are overweight. Overweight and obesity are complex and multifaceted conditions, associated with an increased risk of chronic illness and early mortality. While there are known risk factors, these alone do not fully explain the varying outcomes between individuals. Recently, taste receptors have been proposed to have a role in the risk for obesity. These receptors are expressed throughout the gastrointestinal tract. In this system, they may be involved in modulating dietary intake and metabolic processes. The taste 2 family of receptors (T2Rs) detects bitter compounds. Receptors T2R4 and T2R5 detect (-)-epicatechin (epicatechin), an antioxidant polyphenol, which may have protective effects against obesity. However, the potential role for taste receptors in this association has not been explored. This study assessed whether polymorphisms in TAS2R4 (rs2233998 and rs2234001) and TAS2R5 (rs2227264) were associated with body mass index (BMI). Genotyping (Taqman qPCR assays) was performed on DNA extracted from blood samples (n = 563) from an elderly cohort. Homozygosity for the minor allele of all polymorphisms was significantly associated with a lower BMI in males. The TAS2R4-rs2233998 CC genotype, the TAS2R4-rs2234001 CC genotype and the TAS2R5-rs2227264 TT genotype were associated with lower BMI (2.1, 2.1 and 2.2 units; p = 0.002, 0.003 and 0.001, respectively). Epicatechin intake was not associated with BMI and genotype was not associated with epicatechin intake. This suggests that the association between TAS2R genotype and elevated BMI risk occurs through altered extra-oral responses and not directly via altered epicatechin intake.

Characteristics of Brucella abortus vaccine strain A19 reveals its potential mechanism of attenuated virulence

Brucella vaccination is one of the most important strategies for controlling brucellosis in livestock. The A19 strain was the effective vaccine used to control brucellosis in China. However, the characteristics of physiological and attenuated virulence of the A19 strain are not investigated in detail. In this study, we compared the phenotypic characteristics of the A19 to the wild-type strain S2308. Virulence test showed that the A19 was significantly attenuated at chronic infection stage in infected mouse model. In growth analysis, the A19 exhibited a quick growth at exponential phase and premature at stationary phase. The inflammatory response of macrophages infected by the A19 was detected using TaqMan qPCR assay, indicating that the inflammatory level of the A19-infected macrophages was higher than that of the S2308 infection. Cell death analysis showed that the A19 was not cytotoxic for macrophages. Cell infection showed that the A19 reduced its ability to invade, survive and traffic within host cells, and the intracellular A19 hardly excludes lysosome-associated marker LAMP-1, suggesting that the A19 can’t escape the lysosome degradation within host cells. In further study, the sensitivity test exhibited that the A19 is more sensitive to stress and bactericidal factors than the S2308 strain, Western blot and silver staining analysis exhibited that the A19 has a different expression pattern of OMPs and reduces LPS O-antigen expression relative to the S2308 strain. Those data give us a more detailed understanding about the A19 vaccine strain, which will be beneficial for improvement of current Brucella vaccine and overcoming its defects.

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.

Transcriptomic Profiling of the Adaptive and Innate Immune Responses of Atlantic Salmon to Renibacterium salmoninarum Infection.

Bacterial Kidney Disease (BKD), which is caused by a Gram-positive, intracellular bacterial pathogen (Renibacterium salmoninarum), affects salmonids including Atlantic salmon (Salmo salar). However, the transcriptome response of Atlantic salmon to BKD remained unknown before the current study. We used a 44K salmonid microarray platform to characterise the global gene expression response of Atlantic salmon to BKD. Fish (~54 g) were injected with a dose of R. salmoninarum (H-2 strain, 2 × 108 CFU per fish) or sterile medium (control), and then head kidney samples were collected at 13 days post-infection/injection (dpi). Firstly, infection levels of individuals were determined through quantifying the R. salmoninarum level by RNA-based TaqMan qPCR assays. Thereafter, based on the qPCR results for infection level, fish (n = 5) that showed no (control), higher (H-BKD), or lower (L-BKD) infection level at 13 dpi were subjected to microarray analyses. We identified 6,766 and 7,729 differentially expressed probes in the H-BKD and L-BKD groups, respectively. There were 357 probes responsive to the infection level (H-BKD vs. L-BKD). Several adaptive and innate immune processes were dysregulated in R. salmoninarum-infected Atlantic salmon. Adaptive immune pathways associated with lymphocyte differentiation and activation (e.g., lymphocyte chemotaxis, T-cell activation, and immunoglobulin secretion), as well as antigen-presenting cell functions, were shown to be differentially regulated in response to BKD. The infection level-responsive transcripts were related to several mechanisms such as the JAK-STAT signalling pathway, B-cell differentiation and interleukin-1 responses. Sixty-five microarray-identified transcripts were subjected to qPCR validation, and they showed the same fold-change direction as microarray results. The qPCR-validated transcripts studied herein play putative roles in various immune processes including pathogen recognition (e.g., tlr5), antibacterial activity (e.g., hamp and camp), regulation of immune responses (e.g., tnfrsf11b and socs1), T-/B-cell differentiation (e.g., ccl4, irf1 and ccr5), T-cell functions (e.g., rnf144a, il13ra1b and tnfrsf6b), and antigen-presenting cell functions (e.g., fcgr1). The present study revealed diverse immune mechanisms dysregulated by R. salmoninarum in Atlantic salmon, and enhanced the current understanding of Atlantic salmon response to BKD. The identified biomarker genes can be used for future studies on improving the resistance of Atlantic salmon to BKD.

Development of a Sensitive TaqMan qPCR Assay for Detection and Quantification of Venturia inaequalis in Apple Leaves and Fruit and in Air Samples.

A TaqMan quantitative PCR (qPCR) assay based on the translation elongation factor 1-α gene was developed for the quantification of Venturia inaequalis in leaves and fruits of Malus × domestica and in spore trap samples. The designed primers and hydrolysis probe amplified a specific 86-bp fragment for V. inaequalis. The specificity of the assay was tested using 35 strains of V. inaequalis and 20 different fungal species, including common pathogens of apple and other species of Venturia. The limit of detection was 20 fg, which is lower than a single genome of V. inaequalis. The selectivity of the assay was tested using DNA from three cultivars of Malus × domestica, and no influence on pathogen amplification was found. The assay was also validated for repeatability and reproducibility. With this assay, it was possible to detect and quantify V. inaequalis in four cultivars (Ambrosia, Florina, Golden Delicious, and Mondial Gala) in both symptomatic and asymptomatic leaves and in symptomatic Golden Delicious apple fruit stored for 2 months. Furthermore, the assay was successfully tested on spore trap samples originating from apple orchards. The quantification of the molecular assay when compared with the estimated number of V. inaequalis cells, using an optical microscope, showed a correlation coefficient of 0.8186. The developed technique could be used to detect V. inaequalis in asymptomatic samples without any cross-reaction with other fungal species. Furthermore, to improve the efficacy of disease management with a timely application of fungicides, this assay could be used for the analysis of spore trap samples by using an implemented extraction method.