scholarly journals Simultaneous Detection of North American and European Porcine Reproductive and Respiratory Syndrome Virus Using Real-Time Quantitative Reverse Transcriptase–PCR

2005 ◽  
Vol 17 (2) ◽  
pp. 165-170 ◽  
Author(s):  
Steven B. Kleiboeker ◽  
Susan K. Schommer ◽  
Sang-Myeong Lee ◽  
Sandy Watkins ◽  
Wayne Chittick ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
North American ◽  
Simultaneous Detection ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Field Isolates ◽  
Pcr Assays

Porcine reproductive and respiratory syndrome (PRRS) is 1 of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to the heterogeneity of field isolates as well as the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this disease. In this study, real-time ( TaqMan) reverse transcriptase (RT)–PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual-labeled probes were selected from conserved regions of open-reading frame 7 and the 3'-untranslated region. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 83/83 strains (74 NA; 9 EU) previously isolated by cell culture between 1992 and 2003. The analytical sensitivity of both assays was consistently found to be less than a single TCID50, which corresponded to 5–10 RNA molecules, and was not significantly reduced when the reactions were performed in a multiplex format. When performing multiplex reactions, sensitive detection was possible even when 1 viral RNA concentration was up to 5,000-fold higher than the second. The diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation.

Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus

2014 ◽  
Vol 201 ◽  
pp. 79-85 ◽  
Author(s):  
Michele Drigo ◽  
Giovanni Franzo ◽  
Ilaria Belfanti ◽  
Marco Martini ◽  
Alessandra Mondin ◽  
...  
Keyword(s):  
Real Time ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
End Point ◽  
Pcr Assays

scholarly journals Genetic Diversity of Porcine Reproductive and Respiratory Syndrome Virus and Evaluation of Three One-Step Real-Time RT-PCR Assays in Korea

2021 ◽  
Author(s):  
Go-Eun Shin ◽  
Ji-Young Park ◽  
Kyoung-Ki Lee ◽  
Mi-Kyeong Ko ◽  
Bok-Kyung Ku ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Real Time ◽  
Economic Losses ◽  
Respiratory Syndrome Virus ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
The Real ◽  
One Step ◽  
Pcr Assays

Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV. Methods In this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Results A total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated. Conclusion The results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

scholarly journals Normal breathing releases SARS-CoV-2 into the air

2021 ◽  
Author(s):  
Piero Di Carlo ◽  
Katia Falasca ◽  
Claudio Ucciferri ◽  
Bruna Sinjari ◽  
Eleonora Aruffo ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Pcr Detection ◽  
Viral Rna ◽  
Rt Pcr ◽  
Air Samples ◽  
Reverse Transcriptase Pcr ◽  
Normal Breathing ◽  
Exhaled Air ◽  
Negative Contagion

This study tests the release of SARS-CoV-2 RNA into the air during normal breathing, without any sign of possible risk of contagion such as coughing, sneezing or talking. Five patients underwent oropharyngeal, nasopharyngeal and salivary swabs for real-time reverse transcriptase PCR (RT-PCR) detection of SARS-CoV-2 RNA. Direct SARS-CoV-2 release during normal breathing was also investigated by RT-PCR in air samples collected using a microbiological sampler. Viral RNA was detected in air at 1 cm from the mouth of patients whose oropharyngeal, nasopharyngeal and salivary swabs tested positive for SARS-CoV-2 RNA. In contrast, the viral RNA was not identified in the exhaled air from patients with oropharyngeal, nasopharyngeal and salivary swabs that tested negative. Contagion of SARS-CoV-2 is possible by being very close to the mouth of someone who is infected, asymptomatic and simply breathing.

scholarly journals Immunomagnetic Separation Combined with Real-Time Reverse Transcriptase PCR Assays for Detection of Norovirus in Contaminated Food

2008 ◽  
Vol 74 (13) ◽  
pp. 4226-4230 ◽  
Author(s):  
YoungBin Park ◽  
You-Hee Cho ◽  
YoungMee Jee ◽  
GwangPyo Ko
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Food Borne ◽  
Contaminated Food ◽  
Microbial Contaminants ◽  
Pcr Assays ◽  
Sensitive Method

ABSTRACT We developed an immunomagnetic separation (IMS) technique combined with real-time TaqMan reverse transcriptase PCR (RT-PCR), which allowed detection of norovirus at a level as low as 3 to 7 RT-PCR units from artificially contaminated strawberries. The inoculum recovery rate ranged from 14 to 30%. The data demonstrate that IMS combined with real-time RT-PCR will be useful as a rapid and sensitive method for detecting food-borne microbial contaminants.

Critical Issues in Detecting Viable Listeria monocytogenes Cells by Real-Time Reverse Transcriptase PCR

2012 ◽  
Vol 75 (3) ◽  
pp. 512-517 ◽  
Author(s):  
LINLIN XIAO ◽  
LU ZHANG ◽  
HUA H. WANG
Keyword(s):  
Listeria Monocytogenes ◽  
16S Rrna ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Pcr Primers ◽  
Detection Methods ◽  
Rt Pcr ◽  
Heat Treated ◽  
Pcr Assays ◽  
The Impact

Rapid and specific detection of viable Listeria monocytogenes cells, particularly in processed foods, is a major challenge in the food industry. To assess the suitability of using RNA-based detection methods to detect viable cells, several sets of PCR primers and florescent probes were designed targeting the 16S rRNA, internalin A, and ribosomal protein L4 genes. One-step real-time reverse transcriptase (RT) PCR assays were conducted using RNAs extracted from control and heat-treated L. monocytogenes samples. The cycle threshold values were significantly higher in heat-treated cells than in controls. However, real-time RT-PCR amplification signals were still detected even in samples stored at room temperature for 24 h after lethal treatments, and the intensity of the signals was correlated with the cell population. The 16S rRNA molecules were the most stable of the three targets evaluated, and the impact on detection efficacy of the relative positions of the PCR primers within the target genes was limited under the experimental conditions. These results suggest that real-time RT-PCR assays have advantages over conventional PCR assays for assessing viable L. monocytogenes cells, but the results are affected by the stability of the RNA molecules targeted. These findings could have a major impact on interpretation of RNA-based detection data and gene expression studies.

Development of universal and quadruplex real‐time RT‐PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses

2019 ◽  
Vol 66 (6) ◽  
pp. 2271-2278 ◽  
Author(s):  
Nanhua Chen ◽  
Mengxue Ye ◽  
Yanzhao Xiao ◽  
Shuai Li ◽  
Yucheng Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assays

scholarly journals Recombinant swine beta interferon protects swine alveolar macrophages and MARC-145 cells from infection with Porcine reproductive and respiratory syndrome virus

2007 ◽  
Vol 88 (3) ◽  
pp. 925-931 ◽  
Author(s):  
C. Overend ◽  
R. Mitchell ◽  
D. He ◽  
G. Rompato ◽  
M. J. Grubman ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Real Time ◽  
Alveolar Macrophages ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Beta Interferon ◽  
293 Cells

Swine beta interferon (swIFN-β) produced in HEK 293 cells infected with a recombinant, replication-defective human adenovirus 5 (Ad5) encoding the swIFN-β gene was tested for antiviral activity against Porcine reproductive and respiratory syndrome virus (PRRSV). MARC-145 cells were incubated overnight with dilutions of supernatant fluids from HEK 293 cells infected with Ad5-swIFN-β or with an Ad5 control virus (Ad5-Blue). Treated cells were infected with PRRSV; MARC-145 cells incubated with Ad5-Blue supernatants developed cytopathic effects (CPE), whereas those incubated with swIFN-β showed no CPE. To confirm the antiviral activity of swIFN-β, culture fluids from Ad5-swIFN-β-infected cells were affinity-purified on a Sepharose–anti-swIFN-β matrix, and the resulting fractions exhibited antiviral activity upon infection with PRRSV. The antiviral effects were specific, as they were blocked by mAbs against swIFN-β. Additional cultures of MARC-145 cells treated with swIFN-β-containing supernatants or affinity-purified swIFN-β were infected with PRRSV and tested by real-time RT-PCR for viral RNA in culture supernatants at various times post-inoculation. These experiments confirmed the protective effects of swIFN-β. swIFN-β was also tested for antiviral activity on porcine alveolar macrophages (PAMs) obtained by bronchoalveolar lavage from PRRSV-negative swine. PAMs were treated with dilutions of swIFN-β or Ad5-Blue culture fluids, infected with PRRSV and tested for viral RNA by real-time RT-PCR. The viral load data showed a dose-dependent protection in swIFN-β-treated PAMs, whereas no protection was evident from Ad5-Blue culture fluids. The data demonstrate that swIFN-β protects both MARC-145 cells and PAMs from PRRSV infection.

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