scholarly journals Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.

Plants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 273
Author(s):  
Alfredo Diaz-Lara ◽  
Kristian Stevens ◽  
Vicki Klaassen ◽  
Deborah Golino ◽  
Maher Al Rwahnih
Keyword(s):  
Genetic Diversity ◽  
Real Time ◽  
Germplasm Collection ◽  
Fruit Trees ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Economic Losses ◽  
Rt Pcr ◽  
Pcr Assays ◽  
Prunus Spp

Viruses can cause economic losses in fruit trees, including Prunus spp., by reducing yield and marketable fruit. Given the genetic diversity of viruses, reliable diagnostic methods relying on PCR are critical in determining viral infection in fruit trees. This study evaluated the broad-range detection capacity of currently available real-time RT-PCR assays for Prunus-infecting viruses and developed new assays when current tests were inadequate or absent. Available assays for 15 different viruses were exhaustively evaluated in silico to determine their capacity to detect virus isolates deposited in GenBank. During this evaluation, several isolates deposited since the assay was designed exhibited nucleotide mismatches in relation to the existing assay’s primer sequences. In cases where updating an existing assay was impractical, we performed a redesign with the dual goals of assay compactness and comprehensive inclusion of genetic diversity. The efficiency of each developed assay was determined by a standard curve. To validate the assay designs, we tested them against a comprehensive set of 87 positive and negative Prunus samples independently analyzed by high throughput sequencing. As a result, all the real-time RT-PCR assays described herein successfully detected the different viruses and their corresponding isolates. To further validate the new and updated assays a Prunus germplasm collection was surveyed. The sensitive and reliable detection methods described here will be used for the large-scale pathogen testing required to maintain the highest quality nursery stock.

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.

Development of three duplex real-time RT-PCR assays for the sensitive and rapid detection of a phytoplasma and five viral pathogens affecting stone fruit trees

2020 ◽  
Vol 53 ◽  
pp. 101621
Author(s):  
Polyxeni G. Pappi ◽  
Ioanna Fotiou ◽  
Konstantinos E. Efthimiou ◽  
Nikolaos I. Katis ◽  
Varvara I. Maliogka
Keyword(s):  
Real Time ◽  
Rapid Detection ◽  
Fruit Trees ◽  
Stone Fruit ◽  
Rt Pcr ◽  
Viral Pathogens ◽  
Pcr Assays

scholarly journals Diagnostics of Grapevine fanleaf virus

Biljni lekar ◽  
2021 ◽  
Vol 49 (1) ◽  
pp. 54-64
Author(s):  
Đina Konstantin ◽  
Goran Barać ◽  
Renata Iličić ◽  
Ferenc Bagi
Keyword(s):  
Visual Inspection ◽  
Virus Detection ◽  
Plant Viruses ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Economic Losses ◽  
Rt Pcr ◽  
Grapevine Fanleaf Virus ◽  
Qrt Pcr ◽  
Propagation Material

Plant viruses cause considerable economic losses and are a threat for sustainable agriculture. Due to the multiple possibilities of infection, they have become widespread. The use of healthy propagation material, free of viroids, viruses and bacteria, is an important strategy in disease control in viticulture. The early and accurate detection of plant viruses is an essential component of their control. Due to the widespread of Grapevine fanleaf virus (GFLV) and its devastating potential, various diagnostic methods are being used. GFLV detection methods based on the specificity of the protein cover (ELISA) and nucleic acid-based virus detection methods (RT-PCR, qRT-PCR). Symptoms of viral diseases are often not distinct and can be confused with those of abiotic stresses, so visual inspection is not reliable enough.

scholarly journals Novel Human Astroviruses: Prevalence and Association with Common Enteric Viruses in Undiagnosed Gastroenteritis Cases in Spain

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 585 ◽  
Author(s):  
Diem-Lan Vu ◽  
Aurora Sabrià ◽  
Nuria Aregall ◽  
Kristina Michl ◽  
Virginia Rodriguez Garrido ◽  
...  
Keyword(s):  
Real Time ◽  
Acute Gastroenteritis ◽  
Enteric Viruses ◽  
Epidemiological Studies ◽  
Diagnostic Methods ◽  
Rt Pcr ◽  
Routine Diagnostics ◽  
Human Astroviruses ◽  
Stool Samples ◽  
Pcr Assays

A remarkable percentage of acute gastroenteritis cases remain etiologically undiagnosed. The aim of the study was to determine the prevalence of common and emerging enteric viruses, such as novel human astroviruses, among undiagnosed samples from children with acute gastroenteritis. Epidemiological studies for novel human astroviruses are still scarce. Stool samples collected over two consecutive winter seasons (2016–2017) from children with gastroenteritis in Spain, which were negative for bacteria, rotavirus, and adenovirus by routine diagnostics were screened by real-time RT-PCR assays for the presence of classical and novel astrovirus, rotavirus, norovirus GI and GII, sapovirus, and adenovirus. Overall, 220/384 stool samples (57.3%) were positive for at least one virus. Co-infections were identified in 21% of cases. Among a total of 315 viruses identified, adenovirus was the most prevalent (n = 103), followed by rotavirus (n = 51), sapovirus (n = 50), classical astrovirus (n = 43), novel astroviruses (n = 42), and norovirus (n = 26). Novel astroviruses were present in 13.3% of virus-positive cases. Most novel astroviruses were found in children <2-year-old (30/39 children, 77%, p = 0.01) and were found in co-infection (66%). Only classical astroviruses demonstrated significant differences in the Cq values during mono-infections compared to co-infections. In conclusion, common enteric viruses may be frequently found in children with undiagnosed gastroenteritis, indicating the need to implement more sensitive diagnostic methods. Novel astroviruses circulate in the community and could be the cause of gastroenteritis among young children.

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 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

scholarly journals Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Ute Eberle ◽  
◽  
Clara Wimmer ◽  
Ingrid Huber ◽  
Antonie Neubauer-Juric ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Pcr Assays ◽  
Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

scholarly journals Quantitation of replication of the HCV genome in human livers with end-stage cirrhosis by strand-specific real-time RT-PCR assays: Methods and clinical relevance

2009 ◽  
Vol 81 (9) ◽  
pp. 1569-1575 ◽  
Author(s):  
Lan Lin ◽  
Louis Libbrecht ◽  
Jannick Verbeeck ◽  
Chris Verslype ◽  
Tania Roskams ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Relevance ◽  
Rt Pcr ◽  
Pcr Assays ◽  
Human Livers ◽  
End Stage

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 Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

2009 ◽  
Vol 39 (5) ◽  
pp. 1445-1451 ◽  
Author(s):  
Helena Lage Ferreira ◽  
Fernando Rosado Spilki ◽  
Márcia Mercês Aparecida Bianchi dos Santos ◽  
Renata Servan de Almeida ◽  
Clarice Weis Arns
Keyword(s):  
Real Time ◽  
Comparative Evaluation ◽  
Virus Isolation ◽  
Diagnostic Methods ◽  
N Gene ◽  
Genomic Rna ◽  
Rt Pcr ◽  
Avian Metapneumovirus ◽  
Lower Detection ◽  
Subtype A

Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an established test for the attachment (G) gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3) to 10¹ TCID50 mL-1). The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.

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