scholarly journals Differentiation of PEDV Classical Attenuated Vaccine Strains from Wild-type Strains using One-Step Real-Time Fluorescent Reverse Transcription PCR Assay Targeting ORF1 Nucleotides Deletion Region

2021 ◽  
Author(s):  
Zhilin Wang ◽  
Xuerui Li ◽  
Youjun Shang ◽  
Jinyan Wu ◽  
Zhen Dong ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Economic Losses ◽  
Wild Type ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Attenuated Vaccine ◽  
Reverse Transcription Pcr ◽  
One Step ◽  
Type Strains

Abstract BackgroundPorcine epidemic diarrhea virus (PEDV) is a pathogen causing serious disease and resulting in severe economic losses in the swine industry. In recent years, although China has adopted a large-scale vaccine immunization strategy, many types of PEDV strains, including classical attenuated vaccine strains, have been discovered in the immunized pig herds. Therefore, monitoring the prevalence of different types of PEDV strains is particularly important for the production of pigs and the safety evaluation of related attenuated vaccines MethodsIn the study, a one-step real-time fluorescent reverse transcription PCR (one-step real-time RT-PCR) assay targeting 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains (derived from classical strains) was established, which could effectively distinguish PEDV classical attenuated vaccine strains and wild-type strains. ResultsIn our study, the RNA detection limits for PEDV wild-type strains and classical attenuated vaccine strains were 3.0×103 copies and 3.0×102 copies, respectively. This assay was highly specific for PEDV, with no cross-reactivity for other viruses, causing diarrheal disease. A total of 117 swine fecal samples were analysed by this established real-time RT-PCR assay, indicating that classical attenuated vaccine strains were present in the swine herds in Gansu province, China. Additionally, a pair of primers and two probes of the established assay can be placed in one reaction tube to distinguish PEDV classical attenuated vaccine strains and wild-type strains. ConclusionOur results provided an effective and cheap technology platform for clinical rapid identification testing and epidemiological investigations of PEDV wild-type strains and classical attenuated vaccine strains

scholarly journals Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

2002 ◽  
Vol 68 (3) ◽  
pp. 1351-1357 ◽  
Author(s):  
Camile Pizeta Semighini ◽  
Mozart Marins ◽  
Maria Helena S. Goldman ◽  
Gustavo Henrique Goldman
Keyword(s):  
Quantitative Analysis ◽  
Real Time ◽  
Aspergillus Nidulans ◽  
Abc Transporter ◽  
Reverse Transcription ◽  
Wild Type ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Reverse Transcription Pcr ◽  
Nitroquinoline Oxide

ABSTRACT The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background.

Investigations on the Frequency of Norovirus Contamination of Ready-to-Eat Food Items in Istanbul, Turkey, by Using Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (5) ◽  
pp. 840-843 ◽  
Author(s):  
AYSUN YILMAZ ◽  
KAMIL BOSTAN ◽  
EDA ALTAN ◽  
KARLO MURATOGLU ◽  
NURI TURAN ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Epidemiological Studies ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Food Items ◽  
Reverse Transcription Pcr ◽  
Significant Difference ◽  
Tomato Sample ◽  
Pcr Assays

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

scholarly journals An Integrated Diagnostic Approach for Citrus Exocortis Viroid

2021 ◽  
Author(s):  
Chih-Hsu Lin ◽  
Ting-Hsuan Hung ◽  
I Hu ◽  
Ta-Hsin Ku ◽  
Chun-Yi Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Severe Disease ◽  
Blot Hybridization ◽  
Rt Pcr ◽  
Dot Blot ◽  
Tomato Cultivar ◽  
Dot Blot Hybridization ◽  
Reverse Transcription Pcr ◽  
One Step

Abstract BackgroundCitrus exocortis viroid (CEVd) is a circular single-stranded RNA pathogen consists of around 370 nucleotides and leads to a severe disease showing bark scaling symptom on citrus crops, which leads to yield decrease and economic loss. Since the absence of viroid-encoded proteins, methods for CEVd detection mainly counts on bioassays or nucleic acid-base approaches. In order to validate the CEVd disease, here we developed an integrated diagnostic protocol. MethodsCEVd transcripts were inoculated onto two susceptible cultivars of Solanum lycopersicum L., cv. Rutgers and cv. Double-Fortune, seedings. After inoculation, total RNAs of the two tomato cultivars were extracted to detect CEVd infection by dot blot hybridization, one-step reverse transcription PCR (one-step RT-PCR) and real-time reverse transcription PCR (real-time RT-PCR). In addition, the symptom development of both cultivars was recorded weekly. ResultsThe tomato cultivar Rutgers rather than Double-Fortune or others was selected as a suitable CEVd-indicator plant and the bio-index score was established based on epinasty, vein necrosis, leaf size reduction and stunting symptoms. In addition, the isolate of CEVd that collected from citrus field could rapidly and consistently cause the index symptoms on Rutgers. As expected, CEVd could be specifically and sensitively detected in both tomato and citrus plants by dot-blot hybridization and RT-PCR technologies, including one-step RT-PCR and real-time RT-PCR. Furthermore, we found that the levels of CEVd genomic RNA or CEVd derived small RNAs are correlated to symptom severity. ConclusionsIn this study, we developed an integrated detection method for CEVd and revealed potential underlying viroid-host interactions.

scholarly journals An Improved One-Step Real-Time Reverse Transcription-PCR Assay for Detection of Norovirus

2015 ◽  
Vol 54 (2) ◽  
pp. 497-499 ◽  
Author(s):  
Ilona Glowacka ◽  
Gabrielle Harste ◽  
Jennifer Witthuhn ◽  
Albert Heim
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
One Step

scholarly journals Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay

2011 ◽  
Vol 49 (7) ◽  
pp. 2620-2624 ◽  
Author(s):  
Susan Bennett ◽  
Heli Harvala ◽  
Jeroen Witteveldt ◽  
E. Carol McWilliam Leitch ◽  
Nigel McLeish ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
One Step

scholarly journals One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1560 ◽  
Author(s):  
Rashi Gautam ◽  
Slavica Mijatovic-Rustempasic ◽  
Mathew D. Esona ◽  
Ka Ian Tam ◽  
Osbourne Quaye ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Wild Type ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Group A ◽  
One Step ◽  
Stool Samples ◽  
Pcr Assays ◽  
Type Strains

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

scholarly journals Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays

1998 ◽  
Vol 36 (3) ◽  
pp. 628-633 ◽  
Author(s):  
Gerd Haberhausen ◽  
Judith Pinsl ◽  
Carl-Christoph Kuhn ◽  
Christine Markert-Hahn
Keyword(s):  
Reverse Transcription ◽  
Dynamic Range ◽  
Small Region ◽  
Wild Type ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Standard Curve ◽  
Reverse Transcription Pcr ◽  
Parallel Data ◽  
Rna Fragments

Four different standardization approaches based on a competitive reverse transcription (RT)-PCR assay were compared with a noncompetitive assay based on an external standard curve. Criteria for assessment were accuracy in quantitation, correctness of recovery, sensitivity, dynamic range, reproducibility, throughput, and convenience of sample handling. As a model system, we used the 5′-noncoding region of hepatitis C virus (HCV) for amplification in all quantitative RT-PCRs. A computer program that allowed parallel data processing was developed. Surprisingly, all methods were found suitable for accurate quantitation and comparable with respect to the criterion correctness of recovery. All results differed only by a factor of about 2. The reason for this finding might be that all of our mimics, as well as the wild-type genome of HCV, exhibited exactly the same amplification and hybridization efficacy. Moreover, minimal competition occurred in our experiments over a 5-log dynamic range. A further topic of our investigation was the comparison of two different competitive RNA fragments, mimics, with regard to their suitability as internal standards. One was a heterologous mimic, in which only the primer binding sites were identical to the wild type. The second one was a homologous mimic identical to the wild type except for a small region used for differential hybridization, which was replaced by a permutated sequence of the same length. Both the homologous and heterologous internal mimics were found appropriate for an accurate competitive RT-PCR assay, provided that amplification efficacy, as well as capture efficacy, is proven identical for both analyte and mimic.

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