TaqMan-based real-time PCR method for detection of Yersinia pseudotuberculosis in food.
Ontology highlight
ABSTRACT:
SUBMITTER: Lambertz ST
PROVIDER: S-EPMC2570298 | biostudies-literature | 2008 Oct
REPOSITORIES: biostudies-literature
Publications
TaqMan-based real-time PCR method for detection of Yersinia pseudotuberculosis in food.
Lambertz S Thisted ST Nilsson C C Hallanvuo S S
Applied and environmental microbiology 20080829 20
A sensitive and specific assay for detection of food-borne pathogenic Yersinia pseudotuberculosis was developed. The primer-probe set was designed to target a 157-bp sequence of the chromosomally located gene ail. The complete method, including an internal amplification control, was evaluated for several different food items. ...[more]
Similar Datasets
Project description:The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination.
| S-EPMC2565965 | biostudies-literature
Project description: Not available
| S-EPMC4313340 | biostudies-literature
Project description:So far, only a few cases of immunoglobulin E (IgE)-mediated coconut allergies have been described in the literature. Due to a growing consumption of coconut-containing foods in occidental countries, the number of coconut allergies may also increase. As there is no causative immunotherapy in clinical routine, appropriate food labelling is particularly important, also with regard to cross-contamination, to prevent serious health consequences. The purpose of this study was to develop a DNA-based detection method for coconut (Cocos nucifera). Initially, three sets of coconut-specific primers were designed and tested. A TaqMan™ probe was then developed to identify and quantify coconut by real-time PCR assay. With 27 other plant and animal species, the specificity of the primer/probe system was tested and cross reactivity was excluded. In a dilution series, a limit of detection of 1 pg/µL was determined. Thus, the developed real-time PCR assay is a suitable method to detect coconut in food.
| S-EPMC7143811 | biostudies-literature
Project description:Langat virus (LGTV), one of the members of the tick-borne encephalitis virus (TBEV) complex, was firstly isolated from Ixodes granulatus ticks in Malaysia. However, the prevalence of LGTV in ticks in the region remains unknown. Surveillance for LGTV is therefore important and thus a tool for specific detection of LGTV is needed. In the present study, we developed a real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) for rapid detection of LGTV. Our findings showed that the developed qRT-PCR could detect LGTV at a titre as low as 0.1 FFU/ml. The detection limit of the qRT-PCR assay at 95% probability was 0.28 FFU/ml as determined by probit analysis (p ≤ 0.05). Besides, the designed primers and probe did not amplify ORF of the E genes for some closely related and more pathogenic viruses including TBEV, Louping ill virus, Omsk hemorrhagic fever virus (OHFV), Alkhurma virus (ALKV), Kyasanur Forest Disease virus (KFDV) and Powassan virus (POWV) which showed the acceptable specificity of the developed assay. The sensitivity of the developed method also has been confirmed by determining the LGTV in infected tick cell line as well as LGTV- spiked tick tissues.
| S-EPMC4566139 | biostudies-literature
Project description:Viral canine diarrhea has high morbidity and mortality and is prevalent worldwide, resulting in severe economic and spiritual losses to pet owners. However, diarrhea pathogens have similar clinical symptoms and are difficult to diagnose clinically. Thus, fast and accurate diagnostic methods are of great significance for prevention and accurate treatment. In this study, we developed a one-step multiplex TaqMan probe-based real-time PCR for the differential diagnosis of four viruses causing canine diarrhea including, CPV (Canine Parvovirus), CCoV (Canine Coronavirus), CAstV (Canine Astrovirus), and CaKoV (Canine Kobuviruses). The limit of detection was up to 102 copies/μL and performed well with high sensitivity and specificity. This assay was optimized and used to identify possible antagonistic relationships between viruses. From this, artificial pre-experiments were performed for mixed infections, and a total of 82 canine diarrhea field samples were collected from different animal hospitals in Zhejiang, China to assess the method. The virus prevalence was significantly higher than what previously reported based on RT-PCR (Reverse Transcription-Polymerase Chain Reaction). Taken together, these results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression.
| S-EPMC7286240 | biostudies-literature
Project description:A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of bovine coronavirus (BCoV) RNA in clinical samples is described. The assay is based on TaqMan technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the transmembrane-protein gene of BCoV. The BCoV real-time RT-PCR assay was able to detect the tested BCoV and BCoV-like viruses (canine respiratory coronavirus and bubaline coronavirus), whereas other common viral pathogens of cattle were not recognised by the established oligonucleotide set, thus showing that the test was specific for bovine-like CoVs. The detection limit of the assay was 20 BCoV RNA copies (1-log higher with respect to traditional gel-based RT-PCR) and the reproducibility was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. Two hundred and twenty clinical specimens (92 rectal, 82 nasal and 46 ocular swabs) were subjected to gel-based and real-time RT-PCR. By conventional amplification, 43 rectal, 54 nasal and 34 ocular samples tested positive, whereas the TaqMan assay was able to detect the BCoV nucleic acid in 49 rectal, 60 nasal and 37 ocular swabs. The rapidity and high throughput of the BCoV TaqMan assay makes this method a powerful tool for a sensitive and specific diagnosis of BCoV infection in cattle.
| S-EPMC7112840 | biostudies-literature
Project description:BackgroundDNA genotyping with mutation-specific TaqMan(R) probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations.MethodsMinor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (T(c)) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T(c), followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors.ResultsCOLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors.ConclusionsThe major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.
| S-EPMC2754313 | biostudies-literature
Project description:Porcine circovirus type 2 (PCV2) and the associated disease postweaning multisystemic wasting syndrome (PMWS) have caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. To establish a sensitive, specific assay for the detection and quantitation of PCV2, we designed and synthesized specific primers and a probe in the open reading frame 2. The assay had a wide dynamic range with excellent linearity and reliable reproducibility, and detected between 102 and 1010 copies of the genomic DNA per reaction. The coefficient of variation for Ct values varied from 0.59% to 1.05% in the same assay and from 1.9% to 4.2% in 10 different assays. The assay did not cross-react with porcine circovirus type 1, porcine reproductive and respiratory, porcine epidemic diarrhea, transmissible gastroenteritis of pigs and rotavirus. The limits of detection and quantitation were 10 and 100 copies, respectively. Using the established real-time PCR system, 39 of the 40 samples we tested were detected as positive.
| S-EPMC3023794 | biostudies-literature
Project description:BackgroundMuscovy duck parvovirus (MDPV) causes high mortality and morbidity in Muscovy ducks, with the pathogenesis of the virus still unknown in many respects. Specific MDPV detection is often rife with false positive results because of high identity at the genomic nucleotide level and antigenic similarity with goose parvovirus (GPV). The objective of this study was to develop a sensitive, highly specific, and repeatable TaqMan-based real-time PCR (qPCR) assay for facilitating the molecular detection of MDPV.ResultsThe specific primers and probe were designed based on the conserved regions within MDPVs, but there was a variation in GPVs of the nonstructural (NS) genes after genetic comparison. After the optimization of qPCR conditions, the detection limit of this qPCR assay was 29.7 copies/μl. The assay was highly specific for the detection of MDPV, and no cross-reactivity was observed with other non-targeted duck-derived pathogens. Intra- and inter-assay variability was less than 2.21%, means a high degree of repeatability. The diagnostic applicability of the qPCR assay was proven that MDPV-positive can be found in cloacal swabs samples, Muscovy duck embryos and newly hatched Muscovy ducklings.ConclusionsOur data provided incidents that MDPV could be possible vertically transmitted from breeder Muscovy ducks to Muscovy ducklings. The developed qPCR assay in the study could be a reliable and specific tool for epidemiological surveillance and pathogenesis studies of MDPV.
| S-EPMC6122767 | biostudies-literature
Project description:Traceability of processed food and feed products has been gaining importance due to the impact that those products can have on human/animal health and to the associated economic and legal concerns, often related to adulterations and frauds as it can be the case for meat and milk. Despite mandatory traceability requirements for the analysis of feed composition, few reliable and accurate methods are presently available to enforce the legislative frame and allow the authentication of animal feeds. In this study, nine sensitive and species-specific real-time PCR TaqMan MGB assays are described for plant species detection in animal feed samples. The method is based on selective real-time qPCR (RT-qPCR) amplification of target genes belonging to the alternative oxidase (AOX) gene family. The plant species selected for detection in feed samples were wheat, maize, barley, soybean, rice and sunflower as common components of feeds, and cotton, flax and peanut as possible undesirable contaminants. The obtained results were compared with end-point PCR methodology. The applicability of the AOX TaqMan assays was evaluated through the screening of commercial feed samples, and by the analysis of plant mixtures with known composition. The RT-qPCR methodology allowed the detection of the most abundant species in feeds but also the identification of contaminant species present in lower amounts, down to 1% w/w. AOX-based methodology provides a suitable molecular marker approach to ascertain plant species composition of animal feed samples, thus supporting feed control and enforcement of the feed sector and animal production.
| S-EPMC5749836 | biostudies-literature