scholarly journals Serological and molecular evidence of Brucella species in the rapidly growing pig sector in Kenya

2020 ◽  
Author(s):  
James Miser Akoko ◽  
Roger Pelle ◽  
Velma Kivali ◽  
Esther Schelling ◽  
Gabriel Shirima ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Epidemiological Data ◽  
Molecular Techniques ◽  
Molecular Evidence ◽  
Rt Pcr ◽  
Growing Pig ◽  
Pcr Targeting ◽  
The Rose

Abstract BackgroundBrucellosis is an emerging, yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi, Kenya were screened in parallel, using the Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp.. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 gene and real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes.ResultsA prevalence of 0.57% (n=4/700) was estimated using RBT. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a multiple real-time PCR. ConclusionThe detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs indicate the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

scholarly journals Serological and Molecular Evidence of Brucella Species in the Rapidly Growing Pig Sector in Kenya

2020 ◽  
Author(s):  
James Miser Akoko ◽  
Roger Pelle ◽  
Velma Kivali ◽  
Esther Schelling ◽  
Gabriel Shirima ◽  
...  
Keyword(s):  
Real Time ◽  
Epidemiological Data ◽  
Molecular Techniques ◽  
Molecular Evidence ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Brucella Infection ◽  
Growing Pig ◽  
Pcr Targeting

Abstract Background: Brucellosis is an emerging yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi region, Kenya were screened in parallel, using both Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 geneand real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes. Results: A prevalence of 0.57% (n = 4/700) was estimated using RBT; none of these samples was positive on cELISA. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a real-time multiplex PCR. Conclusion: The detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs confirm the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

scholarly journals Serological and molecular evidence of Brucella species in the rapidly growing pig sector in Kenya

2020 ◽  
Author(s):  
James Miser Akoko ◽  
Roger Pelle ◽  
Velma Kivali ◽  
Esther Schelling ◽  
Gabriel Shirima ◽  
...  
Keyword(s):  
Real Time ◽  
Epidemiological Data ◽  
Molecular Techniques ◽  
Molecular Evidence ◽  
Brucella Species ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Growing Pig ◽  
Pcr Targeting

Abstract Background: Brucellosis is an emerging yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi region, Kenya were screened in parallel, using both Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 gene and real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes.Results: A prevalence of 0.57% (n = 4/700) was estimated using RBT; none of these samples was positive on cELISA. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a real-time multiplex PCR.Conclusion: The detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs confirm the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

scholarly journals Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0248581
Author(s):  
Clyde S. Manuel ◽  
Cassandra Suther ◽  
Matthew D. Moore ◽  
Lee-Ann Jaykus
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Blot Hybridization ◽  
Molecular Techniques ◽  
Rt Pcr ◽  
Dot Blot ◽  
Dot Blot Hybridization ◽  
One Step

Human norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and less frequently, nested real-time PCR. In this study, we conducted a controlled comparison of two published NoV detection assays: a broadly reactive one-step real-time RT-PCR and a two-step nested real-time PCR assay. A 20% human fecal suspension containing a genogroup II human NoV was serially diluted, genome extracted, and subjected to amplification using the two assays compared via PCR Units. Additional amplicon confirmation was performed by dot blot hybridization using digoxigenin (DIG)-labeled oligonucleotide probes. Both assays displayed similar amplification standard curves/amplification efficiencies; however, the nested assay consistently detected one log10 lower virus. Dot blot hybridization improved the detection limit of the nested real-time PCR by one log10 NoV genome copies but impaired the detection limit of the one-step real-time RT-PCR by one log10 NoV genome copies. These results illustrate the complexities in designing and interpreting molecular techniques having a sufficient detection limit to detect low levels of viruses that might be anticipated in contaminated food and environmental samples.

scholarly journals Misidentification of Bordetella bronchiseptica as Bordetella pertussis using a newly described real-time PCR targeting the pertactin gene

2007 ◽  
Vol 56 (12) ◽  
pp. 1608-1610 ◽  
Author(s):  
Karen B. Register ◽  
Tracy L. Nicholson
Keyword(s):  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
Gene Sequence ◽  
Significant Proportion ◽  
Bordetella Bronchiseptica ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Avian Origin ◽  
Pcr Targeting

Recently, a real-time PCR (RT-PCR) assay based on sequence from the gene for pertactin was proposed for identification of Bordetella pertussis. Here, it is reported that the B. pertussis pertactin gene sequence for the region that encompasses the RT-PCR probe and primers is nearly identical to that of many Bordetella bronchiseptica strains of human and avian origin. Additionally, it is demonstrated that such strains are erroneously identified as B. pertussis using the RT-PCR assay. These data suggest that the use of the assay without confirmatory testing may result in erroneous identification of a significant proportion of human isolates of B. bronchiseptica as B. pertussis.

scholarly journals Ultrasensitive detection of Bacillus anthracis by real time PCR targeting a polymorphism in multi-copy 16S rRNA genes and their transcripts

2021 ◽  
Author(s):  
Peter Braun ◽  
Martin Duy-Thanh Nguyen ◽  
Mathias C Walter ◽  
Gregor Grass
Keyword(s):  
16S Rrna ◽  
Bacillus Anthracis ◽  
Real Time ◽  
Real Time Pcr ◽  
Single Copy ◽  
Rrna Genes ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Species Specific ◽  
Pcr Targeting

The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium’s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific SNP present in 2-5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis–specific 16S rRNA gene allele afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with a LoD of 6.3 copies/reaction. In a dilution-series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This will at least provide results equaling the DNA-based implementation if no RNA is present but will be superior even at the lowest residual rRNA concentrations.

scholarly journals Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus

2007 ◽  
Vol 56 (1) ◽  
pp. 56-65 ◽  
Author(s):  
Dobryan M. Tracz ◽  
Paul G. Backhouse ◽  
Adam B. Olson ◽  
Joanne K. McCrea ◽  
Julie A. Walsh ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Rapid Detection ◽  
Vibrio Vulnificus ◽  
Comparative Sequence Analysis ◽  
Molecular Techniques ◽  
Array Technology ◽  
Species Specific ◽  
Pcr Targeting

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.

Transcriptomic profiling of trophoblast fusion using BeWo and JEG-3 cell lines

2019 ◽  
Vol 25 (12) ◽  
pp. 811-824 ◽  
Author(s):  
H Msheik ◽  
S El Hayek ◽  
M Furqan Bari ◽  
J Azar ◽  
W Abou-Kheir ◽  
...  
Keyword(s):  
Cell Line ◽  
Real Time ◽  
Real Time Pcr ◽  
Target Genes ◽  
Bewo Cell ◽  
Rt Pcr ◽  
Trophoblast Differentiation ◽  
Bewo Cells ◽  
Bewo Cell Line

Abstract In human placenta, alteration in trophoblast differentiation has a major impact on placental maintenance and integrity. However, little is known about the mechanisms that control cytotrophoblast fusion. The BeWo cell line is used to study placental function, since it forms syncytium and secretes hormones after treatment with cAMP or forskolin. In contrast, the JEG-3 cell line fails to undergo substantial fusion. Therefore, BeWo and JEG-3 cells were used to identify a set of genes responsible for trophoblast fusion. Cells were treated with forskolin for 48 h to induce fusion. RNA was extracted, hybridised to Affymetrix HuGene ST1.0 arrays and analysed using system biology. Trophoblast differentiation was evaluated by real-time PCR and immunocytochemistry analysis. Moreover, some of the identified genes were validated by real-time PCR and their functional capacity was demonstrated by western blot using phospho-specific antibodies and CRISPR/cas9 knockdown experiments. Our results identified a list of 32 altered genes in fused BeWo cells compared to JEG-3 cells after forskolin treatment. Among these genes, four were validated by RT-PCR, including salt-inducible kinase 1 (SIK1) gene which is specifically upregulated in BeWo cells upon fusion and activated after 2 min with forskolin. Moreover, silencing of SIK1 completely abolished the fusion. Finally, SIK1 was shown to be at the center of many biological and functional processes, suggesting that it might play a role in trophoblast differentiation. In conclusion, this study identified new target genes implicated in trophoblast fusion. More studies are required to investigate the role of these genes in some placental pathology.

scholarly journals Ultrasensitive Detection of Bacillus anthracis by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts

2021 ◽  
Vol 22 (22) ◽  
pp. 12224
Author(s):  
Peter Braun ◽  
Martin Duy-Thanh Nguyen ◽  
Mathias C. Walter ◽  
Gregor Grass
Keyword(s):  
16S Rrna ◽  
Bacillus Anthracis ◽  
Real Time ◽  
Real Time Pcr ◽  
Single Copy ◽  
Rrna Genes ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Species Specific ◽  
Pcr Targeting

The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium’s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific single nucleotide polymorphism (SNP) present in 2–5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real-time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units, and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single-copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis–specific 16S rRNA gene alleles afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with an LoD of 6.3 copies/reaction. In a dilution series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real-time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This can at least provide results equaling the DNA-based implementation if no RNA is present but is superior even at the lowest residual rRNA concentrations.

scholarly journals Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Selene Rubiola ◽  
Tiziana Civera ◽  
Felice Panebianco ◽  
Davide Vercellino ◽  
Francesco Chiesa
Keyword(s):  
18S Rdna ◽  
Inflammatory Myopathy ◽  
Molecular Techniques ◽  
Diaphragm Muscle ◽  
Economic Losses ◽  
Intermediate Hosts ◽  
Slaughter Cattle ◽  
Significant Difference ◽  
Pcr Targeting

Abstract Background Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis. Methods To reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification. Results Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses. Conclusions Our study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis. Graphic Abstract

Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽  
2007 ◽  
Vol 2 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Koray Ergunay ◽  
Gulcin Altinok ◽  
Bora Gurel ◽  
Ahmet Pinar ◽  
Arzu Sungur ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
San Francisco ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Parvovirus B19 ◽  
Etiologic Agent ◽  
Hydrops Fetalis ◽  
Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

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