scholarly journals Epidemiology of Astrovirus, Norovirus and Sapovirus in Greek pig farms indicates high prevalence of Mamastrovirus suggesting the potential need for systematic surveillance

2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Efthymia Stamelou ◽  
Ioannis A. Giantsis ◽  
Konstantinos V. Papageorgiou ◽  
Evanthia Petridou ◽  
Irit Davidson ◽  
...  
Keyword(s):  
Real Time ◽  
Wide Distribution ◽  
Sybr Green ◽  
Rt Pcr ◽  
Swine Farms ◽  
Porcine Sapovirus ◽  
Porcine Astrovirus ◽  
High Prevalence ◽  
Pig Farms ◽  
Surveillance Programs

Abstract Backround Astrovirus, Norovirus and Sapovirus exhibit a wide distribution in swine pig herds worldwide. However, the association of porcine Astrovirus (PAstV), porcine Norovirus (PoNoV) and porcine Sapovirus (PoSaV) with disease in pigs remains uncertain. In this study, we investigated the prevalence of PAstV, PoNoV and PoSaV in Greek pig farms using both conventional RT-PCR and SYBR-Green Real-time RT-PCR in an effort to compare the sensitivity of the two methods. We examined 1400 stool samples of asymptomatic pigs originating from 28 swine farms throughout Greece in pools of five. Results PAstV was detected in all 28 swine farms examined, with an overall prevalence of 267/280 positive pools (95.4%). Porcine Caliciviruses prevalence was found at 36 and 57 out of the 280 examined samples, by the conventional and SYBR-Green Real time RT-PCR, respectively. Sequencing and phylogenetic analysis of the positive samples revealed that the detected PAstV sequences are clustered within PAstV1, 3 and 4 lineages, with PAstV3 being the predominant haplotype (91.2%). Interestingly, sequencing of the Calicivirus positive samples demonstrated the presence of non-target viruses, i.e. Sapovirus, Kobuvirus and Sapelovirus sequences and one sequence highly similar to bat Astrovirus, while no Norovirus sequence was detected. Conclusions The high prevalence of PAstV in Greek pig farms poses a necessity for further investigation of the pathogenicity of this virus and its inclusion in surveillance programs in case that it proves to be important. To our knowledge, this is the first epidemiological study of these viruses in pig farms in Greece.

scholarly journals Design and evaluation of a unique SYBR Green real-time RT-PCR assay for quantification of five major cytokines in cattle, sheep and goats

2015 ◽  
Vol 11 (1) ◽  
pp. 65 ◽  
Author(s):  
Carinne Puech ◽  
Laurence Dedieu ◽  
Isabelle Chantal ◽  
Valérie Rodrigues
Keyword(s):  
Real Time ◽  
Sybr Green ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Sheep And Goats

scholarly journals Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

2018 ◽  
Vol 6 (1) ◽  
pp. 2 ◽  
Author(s):  
David De la Torre ◽  
Claudete Astolfi-Ferreira ◽  
Ruy Chacon ◽  
Antonio Piantino Ferreira
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Sybr Green ◽  
Molecular Techniques ◽  
Economic Losses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Rotavirus A ◽  
Avian Nephritis Virus ◽  
Polymerase Chain

Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.

scholarly journals Self-Sampling of Oropharyngeal Swabs Among Healthcare Workers for Molecular Detection of Respiratory Viruses: A Valuable Approach for Epidemiological Studies and Surveillance Programs

2020 ◽  
Vol 8 ◽  
Author(s):  
Cristina Galli ◽  
Laura Pellegrinelli ◽  
Gabriele Del Castillo ◽  
Giovanni Forni ◽  
Cecilia Eugenia Gandolfi ◽  
...  
Keyword(s):  
Real Time ◽  
Healthcare Workers ◽  
Molecular Detection ◽  
Respiratory Viruses ◽  
Epidemiological Studies ◽  
Influenza Viruses ◽  
Rt Pcr ◽  
Surveillance Programs ◽  
Group 2 ◽  
Group 1

This study aimed at assessing the validity of self-collected (self-sampled) oropharyngeal (OP) swabs among healthcare workers compared to those collected by trained sentinel general practitioners (GP-sampled) from individuals with influenza-like illness (ILI), to be implemented in epidemiological studies and/or surveillance programs of viral pathogens involved in community respiratory infections. In our study, OP swabs were collected from adults (>18 years) with ILI during the 2018–2019 influenza season. Two groups of samples were considered: group 1−131 self-sampled OP swabs collected by healthcare workers after being trained on the sampling procedure; group 2−131 GP-sampled OP swabs collected from outpatients by sentinel GPs operating within the Italian Influenza Surveillance Network. To assess swabbing quality, following RNA extraction, each sample was tested for the presence of the human ribonuclease P gene (RNP) by in-house real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Samples with a cycle threshold (Ct) <35 were considered adequate for further virological analysis. Influenza viruses (IVs), respiratory syncytial virus (RSV), and rhinovirus (RV) genomes were detected by in-house real-time RT-PCR. All samples were positive to RNP detection with Ct <35. The mean Ct value was similar in the two groups (group 1 vs. group 2: 25.93 ± 2.22 vs. 25.46 ± 2.40; p = 0.10). IVs, RSV, and RV positivity rates were 26.7 vs. 52.7% (p < 0.01), 7.6 vs. 9.9% (p = 0.52), and 21.4 vs. 19.9% (p = 0.76), respectively. Self-sampled OP swabs resulted as valid as GP-sampled OP swabs for molecular detection of respiratory viruses. Self-swabbing can thus be a worthwhile strategy for sample collection to implement molecular surveillance of respiratory pathogens and carry out epidemiological studies, easily reaching a larger population size.

Monitoring CML28 mRNA Levels in Patients before and Post HSCT by Real-Time Quantitative RT-PCR.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4478-4478
Author(s):  
Donghua Zhang ◽  
Min Dai ◽  
Hongsheng Zhou ◽  
Yaya Wang ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Mrna Level ◽  
Conditioning Regimen ◽  
Sybr Green ◽  
Sybr Green I ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Standard Samples ◽  
Hematopoietic Stem ◽  
High Level

Abstract A SYBR Green I real-time quantitative RT-PCR method was established for investigating the correlation between CML28 mRNA expressing levels and relapse of leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitorting of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph+ ALL. The sensitivity of the established method was at 10−4 level, with interassay variation and intraassay variation of standard samples both < 10%. The CML28 was highly expressed in AML and CML-BP or AP. In newly diagnosed group, CML28 was (6.58±2.34)×10−2. In pre-conditioning regimen group was (2.19±0.32)×10−2, in group that 1 month after allo-HSCT was (1.35±1.28)×10−2, in group that 3 months after allo-HSCT was (4.57±6.39)×10−3. CML28 can be detected 3months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2×10−2) survived without relapse, the other 2 patients with high level (>2×10−2) relapsed within one year,1 died and1 received the second time allo-HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2×10−2 and relapsed again. CML28 mRNA level was obviously correlated with the development of diseases. Serial quantification of CML28 mRNA levels were necessary for allo-HSCT recipients, and more informative than a single detection. Use of this assay to evaluate MRD in the patients performed allo-HSCT was helpful for predicition of relapse.

scholarly journals Development and evaluation of a SYBR Green real-time RT-PCR assay for detection of avian hepatitis E virus

2015 ◽  
Vol 11 (1) ◽  
Author(s):  
Qin Zhao ◽  
Sha Xie ◽  
Yani Sun ◽  
Yiyang Chen ◽  
Jiming Gao ◽  
...  
Keyword(s):  
Real Time ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Sybr Green ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Avian Hepatitis E Virus
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