scholarly journals Infectious Agents Identified by Real-Time PCR, Serology and Bacteriology in Blood and Peritoneal Exudate Samples of Cows Affected by Parietal Fibrinous Peritonitis after Caesarean Section

2020 ◽  
Vol 7 (3) ◽  
pp. 134
Author(s):  
Salem Djebala ◽  
Julien Evrard ◽  
Fabien Gregoire ◽  
Damien Thiry ◽  
Calixte Bayrou ◽  
...  
Keyword(s):  
Real Time ◽  
Peritoneal Exudate ◽  
Sterile Inflammation ◽  
Infectious Agents ◽  
Positive Serology ◽  
Bacteriological Culture ◽  
E Coli ◽  
Bovine Herpesvirus 4 ◽  
Polymerase Chain ◽  
Herpesvirus 4

The aim of this study was to identify the pathogens potentially involved in parietal fibrinous peritonitis (PFP). PFP is a complication of laparotomy in cattle, characterized by an accumulation of exudate inside a fibrinous capsule. We have studied 72 cases of PFP in Belgian blue cows, confirmed by a standard diagnostic protocol. Blood was collected to evaluate the presence of antibodies for Mycoplasma bovis(M. bovis), Coxiella burnetii(C. burnetii) and Bovine Herpesvirus 4(BoHV4) by enzyme-linked immunosorbent assays. Peritoneal exudate was obtained from the PFP cavity to perform bacteriological culture, and to identify the DNA of M. bovis, C. burnetii and BoHV4 using real time polymerase chain reaction (qPCR). Bacteriological culture was positive in most peritoneal samples (59/72); Trueperella pyogenes (T. pyogenes) (51/72) and Escherichia coli (E. coli) (20/72) were the most frequently identified. For BoHV4, the majority of cows showed positive serology and qPCR (56/72 and 49/72, respectively). Contrariwise, M. bovis (17/72 and 6/72, respectively) and C. burnetii (15/72 and 6/72, respectively) were less frequently detected (p < 0.0001). Our study proves that PFP can no longer be qualified as a sterile inflammation. Moreover, we herein describe the first identification of BoHV4 and C. burnetii in cows affected by PFP.

Detection and Quantitation of Enterohemorrhagic Escherichia coli O157, O111, and O26 in Beef and Bovine Feces by Real-Time Polymerase Chain Reaction†

2002 ◽  
Vol 65 (9) ◽  
pp. 1371-1380 ◽  
Author(s):  
VIJAY K. SHARMA
Keyword(s):  
Real Time ◽  
Shiga Toxin ◽  
Enterohemorrhagic Escherichia Coli ◽  
Specific Primer ◽  
Chain Reaction ◽  
Pcr Assay ◽  
E Coli ◽  
Taqman Probes ◽  
Polymerase Chain ◽  
Pcr Assays

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and certain non-O157 EHEC serotypes (such as O26:H11, O26: NM, O111:H8, and O111:NM) have emerged as significant causes of human disease throughout the world. Important virulence attributes of EHEC are the intimin protein (encoded by the eae gene) and Shiga toxins 1 and 2 (encoded by the stx1 and stx2 genes, respectively). Two sets of real-time polymerase chain reaction (R-PCR) assays were developed for the simultaneous detection and quantitation of EHEC through the monitoring of the presence of the eae and stx genes, and these assays were evaluated. In the eaeR-PCR assay, three sets of primers and TaqMan probes were designed for the amplification and real-time detection of a portion of the eae gene specific to the EHEC O26, O111, and O157 serotypes. In the stxR-PCR assay, two sets of primers and TaqMan probes were used to amplify and detect the stx1 and stx2 genes. DNA prepared from 67 bacterial strains carrying known virulence markers was tested to determine the specificities of the two assays. In the eaeR-PCR assay, eaeO157- and eaeO111-specific primer-probe sets identified only EHEC O157 and O111 strains, respectively. The eaeO26-specific primer-probe set identified all EHEC O26 isolates and some Shiga toxin–negative serotypes of enteropathogenic E. coli and rabbit diarrheagenic E. coli. The stxR-PCR assay was able to identify only those strains carrying either or both of the Shiga toxin–encoding genes. The detection range of both R-PCR assays was linear over DNA concentrations corresponding to 103 to 108 CFU/ml of an EHEC strain. Both assays were able to detect and quantify very low levels (1 to 10 CFU/g of food or feces) of EHEC in feces and ground beef enriched for 16 h in a modified Trypticase soy broth. In conclusion, eae- and stx-based R-PCR assays are reliable and sensitive methods for the rapid screening and specific and quantitative detection of important serotypes of EHEC in cattle and in foods of bovine origin.

scholarly journals Development of a Polymerase Chain Reaction and Restriction Typing Assay for the Diagnosis of Bovine Herpesvirus 1, Bovine Herpesvirus 2, and Bovine Herpesvirus 4 Infections

2002 ◽  
Vol 14 (4) ◽  
pp. 353-356 ◽  
Author(s):  
Luciana De-Giuli ◽  
Simone Magnino ◽  
Pier Giorgio Vigo ◽  
Iris Labalestra ◽  
Massimo Fabbi
Keyword(s):  
Polymerase Chain Reaction ◽  
Restriction Analysis ◽  
Simultaneous Detection ◽  
Bovine Herpesvirus ◽  
Chain Reaction ◽  
Bovine Herpesvirus 1 ◽  
Bovine Herpesvirus 4 ◽  
Herpesvirus 1 ◽  
Polymerase Chain ◽  
Herpesvirus 4

A multiplex polymerase chain reaction (PCR) method coupled with a restriction analysis of PCR products (PCR with restriction fragment length polymorphism) was developed for the simultaneous detection of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections. The specificity, sensitivity, and practical diagnostic applicability of this method were evaluated. This assay may be also adapted to the diagnosis of suid herpesvirus 1 and equine herpesviruses 1 and 3 and could become a powerful diagnostic tool.

scholarly journals Identification of Salmonella spp and serovars Typhimurium, Enteritidis by qPCR

2020 ◽  
pp. 21-31
Author(s):  
N. Rublenko
Keyword(s):  
Real Time ◽  
High Sensitivity ◽  
High Specificity ◽  
Analytical Sensitivity ◽  
Reaction Products ◽  
Salmonella Spp ◽  
Bacterial Dna ◽  
E Coli ◽  
Polymerase Chain ◽  
Primer Sets

This article presents the results of the identification of the Salmonella genus as well as serovars Enteritidis and Typhimurium by a real-time polymerase chain reaction. We constructed three pairs of primers and fluorescent probes to simultaneously identify the Salmonella genus, serovars Enteritidis and Typhimurium in a qPCR. The specificity of the primers was evaluated on Salmonella strains of different serovars from the National Center for Strains of Microorganisms (UNCMS) strains of the State Scientific Control Institute of Biotechnology and Strains of Microorganisms (SSCIBSM) and 46 Salmonella strains isolated from poultry. E. coli ATCC 25922, Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 19112 from UNCMS collection were used to check the specificity of the primers as heterologous samples. Bacterial DNA was extracted using a DNA Sorb B (Amplisens) kit, and realtime PCR was accomplished with the "Real-time PCR kit" (Syntol) on Bio-rad CFX. A series of 10-fold S. Typhimurium and S. Enteritidis DNA dilutions were studied to evaluate the sensitivity of the primers: 10-1-10-5. The analytical sensitivity of primers for detection of the genus Salmonella is: for S. Typhimurium - 0.25 ng/sample (Typhimurium) and S. Enteritidis - 0.27 ng/ sample (Enteritidis). The results of the studies confirmed the specificity of the primer set and the high sensitivity. No hybridization of primers with DNA samples of other bacteria found, in particular, the nonspecific reaction products were absent. The primer sets for the detection of DNA of Enteritidis and Typhimurium serovars also has high specificity. If necessary, this set of primers can be used to perform a multiplex qPCR, that can simultaneously identify bacteria of the Salmonella genus and differentiate Enteritidis and Typhimurium serovars. Keywords: Salmonella, bacteria, polymerasechainreaction, DNA, qPCR.

scholarly journals Rapid and Accurate Detection of Bacteriophage Activity againstEscherichia coliO157:H7 by Propidium Monoazide Real-Time PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Hui Liu ◽  
Yan D. Niu ◽  
Jinquan Li ◽  
Kim Stanford ◽  
Tim A. McAllister
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Propidium Monoazide ◽  
Direct Plating ◽  
E Coli ◽  
Polymerase Chain ◽  
Phage Activity

Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol ofE. colirequire several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intactE. colicells that survive phage exposure.Escherichia coliO157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cellsE. coliO157:H7. Compared to PMA-qPCR, direct plating overestimated (P< 0.01) phage efficacy as cell surface-attached phage particles lysedE. coliO157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol ofE. coliO157:H7.

scholarly journals Evaluation of real-time PCR for quantitative detection of Escherichia coli in beach water

2013 ◽  
Vol 12 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Jason Tszhin Lam ◽  
Edwin Lui ◽  
Simon Chau ◽  
Cathie Show Wu Kueh ◽  
Ying-kit Yung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Limit Of Quantification ◽  
E Coli ◽  
Polymerase Chain ◽  
Beach Water

The current investigation evaluated the use of real-time polymerase chain reaction (PCR) for quantitative detection of Escherichia coli in marine beach water. Densities of E. coli in 263 beach water samples collected from 13 bathing beaches in Hong Kong between November 2008 and December 2009 were determined using both real-time PCR and culture-based methods. Regression analysis showed that these two methods had a significant positive linear relationship with a correlation coefficient (r) of 0.64. Serial dilution of spiked samples indicated that the real-time PCR had a limit of quantification of 25 E. coli colonies in 100 mL water sample. This study showed that the rapid real-time PCR has potential to complement the traditional culture method of assessing fecal pollution in marine beach water.

scholarly journals Presence of infectious agents and co-infections in diarrheic dogs determined with a real-time polymerase chain reaction-based panel

2014 ◽  
Vol 10 (1) ◽  
pp. 23 ◽  
Author(s):  
Aline Gizzi ◽  
Simone Oliveira ◽  
Christian M Leutenegger ◽  
Marko Estrada ◽  
Denise Kozemjakin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Infectious Agents ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals HIV and Chagas Disease: An Evaluation of the Use of Real-Time Quantitative Polymerase Chain Reaction to Measure Levels of Trypanosoma cruzi Parasitemia in HIV Patients in Cochabamba, Bolivia

2021 ◽  
Author(s):  
Melissa J. Reimer-McAtee ◽  
Carolina Mejia ◽  
Taryn Clark ◽  
Jules Terle ◽  
Monica J. Pajuelo ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Trypanosoma Cruzi ◽  
Real Time ◽  
Chagas Disease ◽  
Blood Clot ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Positive Serology ◽  
Polymerase Chain ◽  
Chronic Chagas Disease

This cross-sectional study evaluated epidemiologic characteristics of persons living with HIV (PWH) coinfected with Trypanosoma cruzi in Cochabamba, Bolivia, and estimated T. cruzi parasitemia by real-time quantitative polymerase chain reaction (qPCR) in patients with and without evidence of reactivation by direct microscopy. Thirty-two of the 116 HIV patients evaluated had positive serology for T. cruzi indicative of chronic Chagas disease (27.6%). Sixteen of the 32 (50%) patients with positive serology were positive by quantitative polymerase chain reaction (qPCR), and four of the 32 (12.5%) were positive by direct microscopy. The median parasite load by qPCR in those with CD4+ < 200 was 168 parasites/mL (73-9951) compared with 28.5 parasites/mL (15–1,528) in those with CD4+ ≥ 200 (P = 0.89). There was a significant inverse relationship between the degree of parasitemia estimated by qPCR from blood clot and CD4+ count on the logarithmic scale (rsBC= –0.70, P = 0.007). The correlation between T. cruzi estimated by qPCR+ blood clot and HIV viral load was statistically significant with rsBC = 0.61, P = 0.047. Given the significant mortality of PWH and Chagas reactivation and that 57% of our patients with CD4+ counts < 200 cells/mm3 showed evidence of reactivation, we propose that screening for chronic Chagas disease be considered in PWH in regions endemic for Chagas disease and in the immigrant populations in nonendemic regions. Additionally, our study showed that PWH with advancing immunosuppression have higher levels of estimated parasitemia measured by qPCR and suggests a role for active surveillance for Chagas reactivation with consideration of treatment with antitrypanosomal therapy until immune reconstitution can be achieved.

A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef

2006 ◽  
Vol 52 (10) ◽  
pp. 992-998 ◽  
Author(s):  
Jane Holicka ◽  
Rebecca A Guy ◽  
Anita Kapoor ◽  
David Shepherd ◽  
Paul A Horgen
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Polymerase Chain Reaction Assay ◽  
Ground Beef ◽  
Automatic Process ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

The purpose of this study was to apply our rapid, integrated double enrichment 5′ nuclease real-time polymerase chain reaction assay for the detection of Escherichia coli O157:H7 and evaluate its efficacy. The assay targeted ground beef, an important vehicle in disease epidemiology. The assay reliably determined in 8 h the presence of E. coli O157:H7 in ground beef at the level of 1 colony-forming unit (CFU)/g. The sensitivity and specificity of the assay were compared with that of standard enrichment diagnostic techniques. A correlation of 100% in detection was achieved to the limit of 1 CFU/g. This assay can be used as a rapid, automatic process for identification of E. coli O157:H7 in ground beef or can be integrated with standard culture procedures, resulting in considerable cost and time savings.Key words: real-time PCR, E. coli O157:H7, ground beef, molecular diagnostics, rapid O157:H7 assay.

scholarly journals Studies on the molecular biological peculiarities of bovine herpesvirus 4

2020 ◽  
Vol 23 (4) ◽  
pp. 411-423
Author(s):  
R. Peshev
Keyword(s):  
Phylogenetic Tree ◽  
Bovine Herpesvirus ◽  
Chain Reaction ◽  
Fragment Analysis ◽  
Biological Features ◽  
Bovine Herpesvirus 4 ◽  
European Strain ◽  
American Strain ◽  
Polymerase Chain ◽  
Herpesvirus 4

Studies on the molecular biological features of bovine herpesvirus 4 (BHV 4) strains isolated in Bulgaria have been conducted. Two types of polymerase chain reaction have been developed and applied to confirm the gB and TK genes. A restrictase fragment analysis was performed using various types of restrictase enzymes. The tested Bulgarian strains differed in their restrictase genomic profile from the reference European strain Movar 33/63 and from the American strain DN 599, and were clearly different each from the other. No clear relationship has been established between the restrictase enzyme profiles and the tropism of the isolated viruses. Sequencing of isolated ВHV 4 strains showed homo­logy with the reference European strain Movar 33/63. After construction of the phylogenetic tree, three ВHV 4 strains were at one branch of the phylogenetic tree, while two other strains were at the branch of reference Movar 33/63 strain. Applied molecular biology methods can be successfully used for differentiation and detailed genetic characterisation of the isolated BHV 4 strains.

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