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Journal of Veterinary Diagnostic... Jul 2018Bovine trichomoniasis is a sexually transmitted disease that results in infertility, abortion, and calf age variability. To date, management strategies include testing...
Bovine trichomoniasis is a sexually transmitted disease that results in infertility, abortion, and calf age variability. To date, management strategies include testing for Tritrichomonas foetus and culling of infected males. Challenges associated with testing include cost of culture medium, time and labor burden of sample incubation and processing, and adverse effects of bacterial growth on detection sensitivity. To overcome these challenges, we developed a direct reverse-transcription quantitative real-time PCR (direct RT-qPCR) utilizing smegma, eliminating the use of culture medium. In an analysis of 166 field samples (56 positives and 110 negatives as determined using microscopic reading of cultures as the reference test), the direct RT-qPCR exhibited 100% diagnostic sensitivity and 100% specificity, whereas the currently employed qPCR (culture qPCR), which utilizes cultured samples, exhibited 95% diagnostic sensitivity and 100% specificity. Agreement between direct RT-qPCR and culture qPCR was 98%. Moreover, direct RT-qPCR identified 3 more positive samples and exhibited lower quantification cycle (Cq) values among positives by culture reading than did culture qPCR (direct RT-qPCR Cq range = 14.6-32.3 vs. culture qPCR Cq range = 18.7-37.4). The direct RT-qPCR enables simplified sample collection, elimination of culture medium, faster results, applicability in cows, and lower cost than culture qPCR.
Topics: Animals; Cattle; Cattle Diseases; Female; Male; Polymerase Chain Reaction; Pregnancy; Protozoan Infections, Animal; Real-Time Polymerase Chain Reaction; Smegma; Specimen Handling; Tritrichomonas foetus
PubMed: 29633923
DOI: 10.1177/1040638718767943 -
Journal of Clinical Laboratory Analysis Oct 2022Sexually transmitted infections (STIs) can have serious consequences, and the global STI incidence remains high. However, there is little information on the frequency of...
BACKGROUND
Sexually transmitted infections (STIs) can have serious consequences, and the global STI incidence remains high. However, there is little information on the frequency of STIs with multiple pathogens according to age. Accordingly, we conducted a study to determine the trends of coinfection with sexually transmitted pathogens according to age in the Republic of Korea from 2018 to 2020.
METHODS
From January 2018 to December 2020, 65,191 samples of swab, urine, and other types submitted for STI screening were obtained from U2Bio Co. Ltd. (Seoul, Republic of Korea). Multiplex polymerase chain reaction, a sensitive and rapid method for simultaneous detection of STIs caused by multiple different pathogens, was performed using an AccuPower STI4C-Plex Real-Time PCR kit, AccuPower STI8A-Plex Real-Time PCR kit, and AccuPower STI8B-Plex Real-Time PCR kit with an Exicycler 96 Real-Time Quantitative Thermal Block.
RESULTS
Of the 65,191 samples tested, 35,366 (54.3%) tested positive for one or more sexually transmitted pathogens. The prevalence of coinfections with two or more sexually transmitted pathogens was inversely proportional to age. Furthermore, the rates of coinfection with sexually transmitted pathogens and age distribution differed according to sex and the sexually transmitted pathogen type.
CONCLUSION
This study confirmed that a significant proportion of patients with STIs are coinfected with multiple pathogens. Public health managers could use these results to recognize and prevent STIs according to age.
Topics: Coinfection; Humans; Multiplex Polymerase Chain Reaction; Prevalence; Real-Time Polymerase Chain Reaction; Sexually Transmitted Diseases
PubMed: 36036770
DOI: 10.1002/jcla.24682 -
The Journal of Veterinary Medical... Jun 2016Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective...
Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective therapy or vaccine; thus, the diagnosis and elimination of BLV-infected cattle are the most effective method to eradicate the infection. Clinical veterinarians need a simpler and more rapid method of diagnosing infection, because both nested polymerase chain reaction (PCR) and real-time PCR are labor intensive, time-consuming, and require specialized molecular biology techniques and expensive equipment. In this study, we describe a novel PCR method for amplifying the BLV provirus from whole blood, thus eliminating the need for DNA extraction. Although the sensitivity of PCR directly from whole blood (PCR-DB) samples as measured in bovine blood containing BLV-infected cell lines was lower than that of nested PCR, the PCR-DB technique showed high specificity and reproducibility. Among 225 clinical samples, 49 samples were positive by nested PCR, and 37 samples were positive by PCR-DB. There were no false positive samples; thus, PCR-DB sensitivity and specificity were 75.51% and 100%, respectively. However, the provirus loads of the samples detected by nested PCR and not PCR-DB were quite low. Moreover, PCR-DB also stably amplified the BLV provirus from tumor tissue samples. PCR-DB method exhibited good reproducibility and excellent specificity and is suitable for screening of thousands of cattle, thus serving as a viable alternative to nested PCR and real-time PCR.
Topics: Animals; Cattle; Enzootic Bovine Leukosis; Leukemia Virus, Bovine; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 26911373
DOI: 10.1292/jvms.15-0577 -
The Journal of Veterinary Medical... Jan 2020We performed a clonality analysis using polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) gene rearrangement, specifically with regard to its utility...
We performed a clonality analysis using polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) gene rearrangement, specifically with regard to its utility as a method to diagnose bovine B-cell lymphoma. PCR for IgH gene rearrangement indicated monoclonal proliferation of B-cells in 24 of 35 cattle with B-cell lymphoma. In contrast, PCR for IgH gene rearrangement in lymph nodes and tumor tissues from 65 cattle diagnosed with tumors other than B-cell lymphoma and non-tumors revealed polyclonal population of B-cells. Sensitivity, specificity, positive predictive value, and negative predictive value for PCR for IgH gene rearrangement for bovine B-cell lymphoma were 68.6%, 100%, 100%, and 85.5%, respectively. Clonality analysis using PCR for IgH gene rearrangement may be useful for adjunctive diagnosis of bovine B-cell lymphoma.
Topics: Animals; Cattle; Cattle Diseases; Clone Cells; Gene Rearrangement, B-Lymphocyte; Genes, Immunoglobulin Heavy Chain; Immunoglobulin Heavy Chains; Lymph Nodes; Lymphoma, B-Cell; Neoplasms; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 31801928
DOI: 10.1292/jvms.19-0418 -
Philosophical Transactions of the Royal... Nov 2019Interactions between unicellular eukaryotes and bacteria are difficult to characterize in the environment owing to their large number and inherently microscopic scale....
Interactions between unicellular eukaryotes and bacteria are difficult to characterize in the environment owing to their large number and inherently microscopic scale. Although particular co-occurrences can be recovered through targeted approaches, e.g. single-cell sequencing or fluorescence hybridization, the vast majority of the interactions remain unseen. Here, we discuss Emulsion, Paired Isolation and Concatenation polymerase chain reaction (epicPCR) as a tool to uncover these interactions in very high throughput. Originally developed for taxonomy-to-function linkage in bacterial communities, epicPCR has the potential to recover the complete interaction network in a given environment at single-cell resolution. This approach relies on the encapsulation of protistan single cells in emulsion droplets that can subsequently be gelified into beads. In this way, encapsulated cells can be exposed to lysis reagents and further phylogenetic paired marker amplification. A bacterium that physically co-occurs with the eukaryote will be jointly trapped, and the amplification will generate a concatenated PCR product containing physically coupled taxonomic markers from both partners, creating a link. Further amplification and sequencing enable the construction of an association pattern with statistically verified physical co-occurrences. Here, we discuss the potential, challenges and limitations of epicPCR. We argue that the microscopic scale at which epicPCR operates, the high throughput it delivers and its exploratory nature make it an unparalleled approach to unravel associations between microbes directly from environmental samples. This article is part of a discussion meeting issue 'Single cell ecology'.
Topics: Bacterial Physiological Phenomena; Eukaryota; Microbial Interactions; Polymerase Chain Reaction
PubMed: 31587646
DOI: 10.1098/rstb.2019.0087 -
Scientific Reports Jun 2017We report on a new colorimetric DNA detection method that takes advantage of the power of polymerase chain reaction (PCR) and the simplicity of the classic litmus test....
We report on a new colorimetric DNA detection method that takes advantage of the power of polymerase chain reaction (PCR) and the simplicity of the classic litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in the increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of the bacterium Clostridium difficile that are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients.
Topics: Colorimetry; DNA Replication; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction
PubMed: 28596600
DOI: 10.1038/s41598-017-03009-z -
Thoracic Cancer Nov 2020Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular...
BACKGROUND
Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one-step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10-15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo.
METHODS
The pEGFP-N1-HA plasmid was constructed by one-step PCR and transformation. Cells were transfected with pEGFP-N1-HA and pEGFP-N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA-GFP fusion protein was detected by confocal microscopy.
RESULTS
The pEGFP-N1-HA plasmid was successfully constructed and HA expression in cells.
CONCLUSIONS
Free from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction.
KEY POINTS
Significant findings of the study A method to clone short DNA into plasmids was found. What this study adds Our study provides a flexible and economical option to clone short DNA into plasmids.
Topics: Cloning, Molecular; DNA; Humans; Plasmids; Polymerase Chain Reaction
PubMed: 33015950
DOI: 10.1111/1759-7714.13660 -
Veterinary Parasitology Apr 2021Cytauxzoonosis is a tick-borne disease of domestic cats with high mortality and narrow therapeutic window, particularly in the southcentral and southeastern United...
Cytauxzoonosis is a tick-borne disease of domestic cats with high mortality and narrow therapeutic window, particularly in the southcentral and southeastern United States. The causative agent is the apicomplexan protozoal parasite Cytauxzoon felis and is primarily transmitted by Amblyomma americanum, the lone star tick. Currently there is no vaccine available to prevent cytauxzoonosis and treatment is often ineffective if not initiated early enough in the course of disease. Early diagnosis and therapeutic intervention are therefore crucial for the survival of infected cats. Several methods are available for diagnosis of cytauxzoonosis, with PCR being the most sensitive. However, current PCR assays, which employ double-stranded DNA intercalating dyes to detect C. felis infection, have inherent limitations such as the potential for false positive detection of non-specific amplification products and inability to provide absolute quantification of parasite load. The objective of this study was to develop a probe-based droplet digital PCR (ddPCR) assay capable of detection and quantification of C. felis load over time and during treatment. The C. felis ddPCR assay was able to (i) reliably detect and quantify C. felis DNA in clinical blood samples from cats with acute cytauxzoonosis and (ii) monitor clinical parasite load in response to anti-protozoal treatment through absolute quantification of C. felis DNA over time. When tested on blood samples from cats with experimental C. felis infection, the assay was able to detect infection in cats as early as 24 h prior to the development of clinical signs. In addition, we demonstrate that this probe-based design can be utilized in traditional real-time PCR systems, with similar detection capabilities as compared to ddPCR. The C. felis probe-based ddPCR was also able to detect infection in samples with lower parasite loads when compared to existing nested PCR assays, although these results were not significant due to small sample size. To the author's knowledge, this is the first reported probe-based ddPCR assay to detect Cytauxzoon felis infection in domestic cats.
Topics: Animals; Cat Diseases; Cats; DNA, Protozoan; Ixodidae; Piroplasmida; Polymerase Chain Reaction; Protozoan Infections, Animal; Tick-Borne Diseases
PubMed: 33765571
DOI: 10.1016/j.vetpar.2021.109413 -
Analytical Chemistry Feb 2021Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic...
Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic purposes. Nonetheless, multiplexed PCR-based detection schemes have hitherto been largely limited by technical challenges associated with nonspecific interactions and other limitations inherent to traditional fluorescence-based assays. Here, we describe a novel strategy for multiplexed PCR-based analysis called Ligation-eNabled fluorescence-Coding PCR (LiNC PCR) that exponentially enhances the multiplexing capability of standard fluorescence-based PCR assays. The technique relies upon a simple, preliminary ligation reaction in which target DNA sequences are converted to PCR template molecules with distinct endpoint fluorescence signatures. Universal TaqMan probes are used to create target-specific multicolor fluorescence signals that can be readily decoded to identify amplified targets of interest. We demonstrate the LiNC PCR technique by implementing a two-color-based assay for detection of 10 ovarian cancer epigenetic biomarkers at analytical sensitivities as low as 60 template molecules with no detectable target cross-talk. Overall, LiNC PCR provides a simple and inexpensive method for achieving high-dimensional multiplexing that can be implemented in manifold molecular diagnostic applications.
Topics: Base Sequence; Fluorescence; Nucleic Acids; Polymerase Chain Reaction
PubMed: 33427441
DOI: 10.1021/acs.analchem.0c04221 -
Archives of Virology Jan 2019The red-crowned crane is one of the rarest crane species, and its population is decreasing due to loss of habitat, poisoning, and infections. Using a viral metagenomics...
The red-crowned crane is one of the rarest crane species, and its population is decreasing due to loss of habitat, poisoning, and infections. Using a viral metagenomics approach, we analyzed the virome of feces from wild and captive red-crowned cranes, which were pooled separately. Vertebrate viruses belonging to the families Picornaviridae, Parvoviridae, Circoviridae, and Caliciviridae were detected. Among the members of the family Picornaviridae, we found three that appear to represent new genera. Six nearly complete genomes from members of the family Parvoviridae were also obtained, including four new members of the proposed genus "Chapparvovirus", and two members of the genus Aveparvovirus. Six small circular DNA genomes were also characterized. One nearly complete genome showing a low level of sequence identity to caliciviruses was also characterized. Numerous viruses believed to infect insects, plants, and crustaceans were also identified, which were probably derived from the diet of red-crowned cranes. This study increases our understanding of the enteric virome of red-crowned cranes and provides a baseline for comparison to those of other birds or following disease outbreaks.
Topics: Animals; Birds; Feces; Genome, Viral; Metagenome; Phylogeny; Polymerase Chain Reaction; Viruses
PubMed: 30225519
DOI: 10.1007/s00705-018-4037-x