scholarly journals Probe-Based Real-Time qPCR Assays for a Reliable Differentiation of Capripox Virus Species

2021 ◽  
Vol 9 (4) ◽  
pp. 765
Author(s):  
Janika Wolff ◽  
Martin Beer ◽  
Bernd Hoffmann
Keyword(s):  
Real Time ◽  
Animal Health ◽  
Diagnostic Methods ◽  
Virus Species ◽  
Time Saving ◽  
Robust Detection ◽  
Highly Sensitive ◽  
Reliable Differentiation ◽  
World Organization ◽  
Real Time Qpcr

Outbreaks of the three capripox virus species, namely lumpy skin disease virus, sheeppox virus, and goatpox virus, severely affect animal health and both national and international economies. Therefore, the World Organization for Animal Health (OIE) classified them as notifiable diseases. Until now, discrimination of capripox virus species was possible by using different conventional PCR protocols. However, more sophisticated probe-based real-time qPCR systems addressing this issue are, to our knowledge, still missing. In the present study, we developed several duplex qPCR assays consisting of different types of fluorescence-labelled probes that are highly sensitive and show a high analytical specificity. Finally, our assays were combined with already published diagnostic methods to a diagnostic workflow that enables time-saving, reliable, and robust detection, differentiation, and characterization of capripox virus isolates.

The role of the mussel Mytilus spp. in the transmission of ostreid herpesvirus-1 microVar

Parasitology ◽  
2017 ◽  
Vol 145 (8) ◽  
pp. 1095-1104 ◽  
Author(s):  
A. J. O’ Reilly ◽  
C. Laide ◽  
A Maloy ◽  
S. Hutton ◽  
B. Bookelaar ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Animal Health ◽  
Viral Transmission ◽  
Diagnostic Methods ◽  
The Pacific ◽  
Herpesvirus 1 ◽  
World Organization ◽  
Ostreid Herpesvirus 1 ◽  
The Impact

AbstractThe Pacific oyster Crassostrea gigas contributes significantly to global aquaculture; however, C. gigas culture has been affected by ostreid herpesvirus-1 (OsHV-1) and variants. The dynamics of how the virus maintains itself at culture sites is unclear and the role of carriers, reservoirs or hosts is unknown. Both wild and cultured mussels Mytilus spp. (Mytilus edulis, Mytilus galloprovincialis and hybrids) are commonly found at C. gigas culture sites. The objective of this study was to investigate if Mytilus spp. can harbour the virus and if viral transmission can occur between mussels and oysters. Mytilus spp. living at oyster trestles, 400–500 m higher up the shore from the trestles and up to 26 km at non-culture sites were screened for OsHV-1 and variants by all the World Organization for Animal Health (OIE) recommended diagnostic methods including polymerase chain reaction (PCR), quantitative PCR (qPCR), histology, in situ hybridization and confirmation using direct sequencing. The particular primers that target OsHV-1 and variants, including OsHV-1 microVar (μVar), were used in the PCR and qPCR. OsHV-1 μVar was detected in wild Mytilus spp. at C. gigas culture sites and more significantly the virus was detected in mussels at non-culture sites. Cohabitation of exposed wild mussels and naïve C. gigas resulted in viral transmission after 14 days, under an elevated temperature regime. These results indicate that mussels can harbour OsHV-1 μVar; however, the impact of OsHV-1 μVar on Mytilus spp. requires further investigation.

scholarly journals Laboratory diagnostics of selected feline respiratory pathogens and their prevalence in the Czech Republic

2019 ◽  
Vol 64 (No. 1) ◽  
pp. 25-32
Author(s):  
D. Lobova ◽  
V. Kleinova ◽  
J. Konvalinova ◽  
P. Cerna ◽  
D. Molinkova
Keyword(s):  
Czech Republic ◽  
Real Time ◽  
Respiratory Infections ◽  
Animal Health ◽  
Diagnostic Methods ◽  
Feline Calicivirus ◽  
The Czech Republic ◽  
Pcr Multiplex ◽  
The Difference ◽  
One Year

Respiratory problems in cats have a multifactorial character. Therapy without the detection of pathogen is often ineffective. Our study was therefore focused on the detection of important feline respiratory bacterial pathogens such as Mycoplasma felis, Chlamydia felis and Bordetella bronchiseptica and viral pathogens such as Felid alphaherpesvirus-1 and feline calicivirus. The goal of this study was to map the occurrence of these pathogens in cat populations in the Czech Republic with the aim of introducing rapid and highly sensitive methods into routine diagnostics and to provide consulting services to animal health professionals based on the acquired data. A total of 218 cats were investigated in the study: 69 were outdoor and 149 were indoor cats. Three groups of animals were compared: up to one year of age (60 cats), one to three years of age (68 cats) and more than three years of age (90 cats). Samples were taken from conjunctiva and/or the oropharynx. Samples originated from cats with various forms of respiratory disease or from healthy cats from different parts of the Czech Republic. Real-Time RT-PCR, multiplex Real-Time PCR, nested PCR and sequencing analyses were performed. Outdoor cats were infected more often (84 detected pathogens in 69 cats) than indoor cats (110 detected infections in 149 cats). More than one pathogen was detected in a total of 38 cats, and six cats were infected with more than two pathogens. The difference was statistically significant in the case of co-infections, but not for mono-infections (P < 0.05). Kittens and young adults up to the age of one year were the most common reservoirs of respiratory infections (only 19 cats out of 60 were negative and positive cats often harboured coinfections). The difference in age groups were not statistically significant (P > 0.05). Concerning the site of the sampling, feline calicivirus, M. felis and B. bronchiseptica were detected more often from oropharynx than from conjunctival swabs. M. felis was slightly more common in clinically diseased animals (39.6%) than in healthy ones (26.1%). The obtained results reveal the frequency of individual pathogens and their co-infections in cats kept on the territory of the Czech Republic, data which can be used to make the treatment of respiratory infections and breeding measures more effective. Therefore, the diagnostic methods are now available to veterinary surgeons with the possibility of consultation and discussion of the results.

scholarly journals Evaluation of six TaqMan RT-rtPCR kits on two thermocyclers for the reliable detection of rabies virus RNA

2018 ◽  
Vol 31 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Evelyne Picard-Meyer ◽  
Carine Peytavin de Garam ◽  
Jean Luc Schereffer ◽  
Emmanuelle Robardet ◽  
Florence Cliquet
Keyword(s):  
Real Time ◽  
Rabies Virus ◽  
Fluorescent Antibody ◽  
Animal Health ◽  
Antibody Test ◽  
Virus Rna ◽  
Field Samples ◽  
Rapid Amplification ◽  
World Organization ◽  
Positive Results

Rabies is diagnosed postmortem in animals, based on tests prescribed by the World Organization for Animal Health (OIE), such as the fluorescent antibody test, the direct rapid immunohistochemistry test, or pan-lyssavirus PCR assays. Several reverse-transcription real-time PCR (RT-rtPCR) methods have been developed and validated for rapid and accurate detection of lyssaviruses. We evaluated the performance of 6 TaqMan RT-rtPCR kits using different commercial master mixes and 2 real-time thermocyclers. Changing the master mix overall did not influence the TaqMan RT-rtPCR performance, regardless of the thermocycler used. The limits of detection at the 95% confidence level were 18.1–25.8 copies/µL for the Rotor-Gene Q MDx thermocycler and 16.7–21.5 for the Mx3005P thermocycler. Excellent repeatability was demonstrated for rabies virus (RABV) RNA samples of 100, 50, and 25 copies/µL regardless of the thermocycler used. RABV field samples ( n = 35) isolated worldwide gave positive results using the most efficient of the 6 kits tested, with a copy number of 6.03 × 102 to 6.78 × 107 RNA copies per reaction. The TaqMan RT-rtPCR assay provides sensitive and rapid amplification of RABV RNA.

scholarly journals MODERN LABORATORY DIAGNOSTIC METHODS IN VETERINARY MEDICINE AND THE POSSIBILITY OF ITS APPLICATION

2010 ◽  
Vol 3 (1) ◽  
pp. 39-61
Author(s):  
Tamaš Petrović ◽  
Maja Velhner ◽  
Jelena Petrović ◽  
Igor Stojanov ◽  
Živoslav Grgić ◽  
...  
Keyword(s):  
Veterinary Medicine ◽  
Real Time ◽  
Virus Detection ◽  
Diagnostic Methods ◽  
Molecular Methods ◽  
Molecular Diagnostic ◽  
Rt Pcr ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Causative Agents

One of the greatest challenges of modern laboratory diagnosis is the selection of methods for fast, highly sensitive and specific detection of the infective agents. This challenge is present also in molecular diagnosing of causative agents in veterinary medicine. An example of molecular detection, the application of molecular diagnostic methods and procedures in the epizootiology of some viral infections is presented in the paper. The molecular methods play an important role in virus detection and surveillance. Out of a large number of molecular methods most frequently used are classical gel-based PCR (PCR, RT-PCR and nested PCR) and real-time PCR or RT-PCR techniques. Due to highly specificity and sensitivity these methods have been introduced as internationally recognized methods for virus detection in clinical materials. The advantage of the aforementioned molecular methods is that they are very fast and highly sensitive, and able to analyse a high number of samples. The obtained results may be used in molecular epizootiology, possibly for differentiation of filed isolates and vaccine virus strains, and for examining the samples not suitable for virus isolation. Alongside, these methods provide accurate quantification of viral particles in sample material. The detection with high sensitivity and specificity is of utmost importance in detection and typisation of the agents of highly contagious diseases and zoonoses. An example of rapid detection and sensitivity of RT-PCR and real-time RT-PCR in detection and characterization of avian influenza virus in clinical material as well as BVD virus in native bull semen is presented. Besides, molecular methods may be used for other purposes. Genome fragments amplified by PCR and RT-PCR, may be sequenced and used for classification, i.e. for virus isolate genotypisation. The results obtained in this way may be used for basic molecular research in epizootiology, that will point on the source of infections, their correlation and the prevalence of the causative agents what can help in finding the answer to the question why the diseases have occurred and what are the perspective for diseases outcome in the future. Also, these examination may help in determining pathogenicity, virulence and the spread of the pathogen agents. These information are of immeasurable importance for all the procedures in disease prevention and control. The possibilities of sequencing, molecular typisation and epizootiology of BVD virus and CSF virus isolated in Vojvodina in the last years are given as an example how this method can be used.

Intestinal parasitic infections in an industrialized country; a new focus on children with better DNA-based diagnostics

Parasitology ◽  
2011 ◽  
Vol 138 (12) ◽  
pp. 1492-1498 ◽  
Author(s):  
JACO J. VERWEIJ ◽  
LISETTE VAN LIESHOUT
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Laboratory ◽  
Intestinal Parasites ◽  
Diagnostic Methods ◽  
Parasitic Infections ◽  
Detection Rates ◽  
Highly Sensitive ◽  
Faecal Samples ◽  
Gastrointestinal Complaints

SUMMARYIn recent years, the isolation of parasitic DNA from faecal samples and PCR techniques, have been improved and simplified. Moreover, the introduction of real-time PCR has made it possible to multiplex different targets into one reaction. These new technical possibilities make it feasible to introduce PCR with its unsurpassed sensitivity and specificity in a routine laboratory setting for the diagnosis of intestinal parasites. Detection rates of the parasitic infections included in the PCR are increased significantly compared with microscopy. Molecular diagnostics, especially in children, reveal a possible cause of the gastrointestinal complaints in many more cases compared with conventional methods. Usually in GP patients no other pathogenic parasites are detected using microscopy and in the returning travellers additional parasites are found with microscopy in a minority of cases only. Multiplex real-time PCR offers a highly sensitive and specific diagnostic alternative for labour intensive microscopy in clinical laboratory practice. Additional diagnostic methods for the detection of parasitic infections that are not included as PCR target can be limited to a selected group of patients.

DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

2019 ◽  
pp. 60-66
Author(s):  
Viet Quynh Tram Ngo ◽  
Thi Ti Na Nguyen ◽  
Hoang Bach Nguyen ◽  
Thi Tuyet Ngoc Tran ◽  
Thi Nam Lien Nguyen ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Type B ◽  
E Coli ◽  
Pcr Assays ◽  
Set Up ◽  
Etiological Agents ◽  
Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

Detection by real-time quaking-induced conversion (RT-QuIC), ELISA, and IHC of chronic wasting disease prion in lymph nodes from Pennsylvania white-tailed deer with specific PRNP genotypes

2021 ◽  
pp. 104063872110214
Author(s):  
Deepanker Tewari ◽  
David Steward ◽  
Melinda Fasnacht ◽  
Julia Livengood
Keyword(s):  
Real Time ◽  
Disease Susceptibility ◽  
Chronic Wasting Disease ◽  
Low Frequency ◽  
The United States ◽  
Detection Methods ◽  
Spongiform Encephalopathy ◽  
Wasting Disease ◽  
Diagnostic Laboratory ◽  
Highly Sensitive

Chronic wasting disease (CWD) is a prion-mediated, transmissible disease of cervids, including deer ( Odocoileus spp.), which is characterized by spongiform encephalopathy and death of the prion-infected animals. Official surveillance in the United States using immunohistochemistry (IHC) and ELISA entails the laborious collection of lymphoid and/or brainstem tissue after death. New, highly sensitive prion detection methods, such as real-time quaking-induced conversion (RT-QuIC), have shown promise in detecting abnormal prions from both antemortem and postmortem specimens. We compared RT-QuIC with ELISA and IHC for CWD detection utilizing deer retropharyngeal lymph node (RLN) tissues in a diagnostic laboratory setting. The RLNs were collected postmortem from hunter-harvested animals. RT-QuIC showed 100% sensitivity and specificity for 50 deer RLN (35 positive by both IHC and ELISA, 15 negative) included in our study. All deer were also genotyped for PRNP polymorphism. Most deer were homozygous at codons 95, 96, 116, and 226 (QQ/GG/AA/QQ genotype, with frequency 0.86), which are the codons implicated in disease susceptibility. Heterozygosity was noticed in Pennsylvania deer, albeit at a very low frequency, for codons 95GS (0.06) and 96QH (0.08), but deer with these genotypes were still found to be CWD prion-infected.

scholarly journals Tick-borne zoonoses and commonly used diagnostic methods in human and veterinary medicine

2021 ◽  
Author(s):  
Andrea Springer ◽  
Antje Glass ◽  
Julia Probst ◽  
Christina Strube
Keyword(s):  
Veterinary Medicine ◽  
Diagnostic Tests ◽  
One Health ◽  
Animal Health ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Diagnostic Tools ◽  
Zoonotic Pathogens ◽  
Health Concept ◽  
The One

AbstractAround the world, human health and animal health are closely linked in terms of the One Health concept by ticks acting as vectors for zoonotic pathogens. Animals do not only maintain tick cycles but can either be clinically affected by the same tick-borne pathogens as humans and/or play a role as reservoirs or sentinel pathogen hosts. However, the relevance of different tick-borne diseases (TBDs) may vary in human vs. veterinary medicine, which is consequently reflected by the availability of human vs. veterinary diagnostic tests. Yet, as TBDs gain importance in both fields and rare zoonotic pathogens, such as Babesia spp., are increasingly identified as causes of human disease, a One Health approach regarding development of new diagnostic tools may lead to synergistic benefits. This review gives an overview on zoonotic protozoan, bacterial and viral tick-borne pathogens worldwide, discusses commonly used diagnostic techniques for TBDs, and compares commercial availability of diagnostic tests for humans vs. domestic animals, using Germany as an example, with the aim of highlighting existing gaps and opportunities for collaboration in a One Health framework.

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