Wuhan coronavirus (2019-nCoV) real-time RT-PCR N gene 2020 (Wuhan-N) v1 (protocols.io.ba86ihze)

protocols.io ◽  
2020 ◽  
Author(s):  
Judy A ◽  
Ian Mackay
Keyword(s):  
Real Time ◽  
N Gene ◽  
Rt Pcr

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.

scholarly journals Development of real-time RT-PCR for N2 subtype avian influenza RNA-virus detection

2020 ◽  
pp. 186-192
Author(s):  
P. B. Akshalova ◽  
A. V. Andriyasov ◽  
L. O. Scherbakova ◽  
S. N. Kolosov ◽  
N. G. Zinyakov ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Real Time ◽  
Disease Virus ◽  
Virus Detection ◽  
N Gene ◽  
Oligonucleotide Primer ◽  
Rt Pcr ◽  
Polymerase Chain Reaction Condition ◽  
Condition Optimization ◽  
Virus Isolates

Currently, N2 subtype avian influenza (AI) virus actively circulates in domestic and wild bird populations and is regularly detected in China, other Asian countries and Russia, particularly in combination with H9 hemagglutinin. Therefore, a method for rapid detection of the said infectious agent is urgently required. Data on oligonucleotide primer selection and reverse transcription real-time polymerase chain reaction condition optimization for N2 AI virus detection are presented in the paper. Modified primers and probe proposed by B. Hoffmann in 2006 as well as original primers and probes with the viruses available in the Laboratory working collection and selected during testing were assessed for N2 neuraminidase gene fragment amplification. Optimal concentrations of real-time RT-PCR master mix components and temperature-time mode were determined. Various combinations of primers were tested against ten N2 avian influenza virus isolates that genetically differed from each other in N gene. Nine viruses were isolated from birds in the Russian Federation regions and classified to different genetic groups. The real-time RT-PCR assay was tested for its specificity using AI virus isolates of different neuraminidase subtypes (H5N8, H3N6, H4N6, H5N1, H10N7) as well as samples containing other RNA-viruses: Newcastle disease virus, infectious bronchitis virus and infectious bursal disease virus. As a result of the testing, real-time RT-PCR conditions providing high sensitivity and specificity of the assay were selected and optimized.

CONSIDERATIONS ON SARS-COV-2 DIAGNOSIS IN THE LABORATORY OF UNIVERSITY EMERGENCY CLINICAL HOSPITAL OF CONSTANTA

2021 ◽  
Author(s):  
Ramona Stoicescu ◽  
Razvan-Alexandru Stoicescu ◽  
Codrin Gheorghe ◽  
Adina Honcea ◽  
Iulian Bratu
Keyword(s):  
Real Time ◽  
Open Reading Frames ◽  
Nucleic Acid Amplification ◽  
Structural Proteins ◽  
N Gene ◽  
Rt Pcr ◽  
Reading Frame ◽  
Clinical Hospital ◽  
The University ◽  
Hospital Wards

Coronaviruses are members of the Coronaviridae family. They are enveloped, non-segmented, positive-sense, single-stranded RNA viruses. Their genome size is about 30 kilobases (kb) which consist, at the 5’ end, of non-structural open reading frames (ORFs: ORF1a, ORF 1b) which code for 16 non structural proteins, and at the 3’ end the genes which code for four structural proteins including membrane (M), envelope (E), spike (S), and nucleocapsid (N) proteins. Due to the rapid spread of COVID-19, a reliable detection method is needed for patient diagnosis especially in the early stages of the disease. WHO has recommended nucleic acid amplification tests such as real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay detects three SARS-CoV-2 RNA targets: the envelope (E) gene, the nucleocapsid (N) gene and a region of the open reading frame (ORF1) of the RNA-dependent RNA polymerase (RdRp) gene from SARS-CoV-2 virus isolate Wuhan-Hu-1. Our study was made in the first 3 months of the year 2021 using the real-time RT PCR results obtained in the Cellular Biology ward of the University Emergency Clinical Hospital. In our lab we are testing the inpatients from the hospital wards (Neurology, Pediatrics, Surgery, Internal medicine, ICU, Cardiology, etc.); we are also testing the outpatients from Dialysis and Oncology, 2 days prior to their therapy; we also test the health care personnel. The number of tests we performed was: in January 1456, with 399 positive results (27.4%), 33 deaths; in February 1273 tests, 221 positive (17.36%), 16 deaths; in March 1471 tests, 373 positive (25.36%), 37 deceased.

CORRELATION OF PROGNOSIS OF COVID POSITIVE PATIENTS BETWEEN REAL TIME RT-PCR AND CT-VALUES OF E. GENE, N.GENE, RDRP GENE

2021 ◽  
pp. 1-3
Author(s):  
Sreyoshi Som ◽  
Indraneel Dasgupta
Keyword(s):  
Real Time ◽  
Virus Disease ◽  
The Other ◽  
Rdrp Gene ◽  
N Gene ◽  
Absolute Amount ◽  
Target Sequence ◽  
Rt Pcr ◽  
E Gene ◽  
Corona Virus

INTRODUCTION: COVID-19 – Corona virus disease-19 – A respiratory illness caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), colloquially referred to as coronavirus. Real-time PCR is conducted to quantify the absolute amount of target sequence or to compare relative amount of a target sequence among samples. This technique monitors amplication of the target in real time via a target specic uorescent signal emitted during amplication. During most real times PCR, a considerable amount of background uorescence occurs. Inspite of this fact PCR uorescent dyes and probes should be sequence specic. This CT value or cycle threshold value in a real time RT- PCR is dened as the number of cycles required for the uorescent signal to cross the threshold i.e., exceeds background level). This is inversely proportional to the amount of target nuclei in the sample. i.e., lower the CTlevel the greater the amount of target nuclei in the sample. AIM: To explore the association of the mutation pattern of covid-19 genetic variation with the severity and fatality of patients with covid-19 viral illness. Factors considered are Age, Sex, and Duration of hospital stay and outcome of patient. MATERIALS AND METHODS: The study was a comparative, correlation, retrospective, lab values based study. All covid+ve patients tested at Peerless hospital, Kolkata by RT-PCR on admission. It is imperative to choose reference genes whose expression levels are not expected to change during our experiment. Common housekeeping genes include actin, alpha-tubuliun, gapdh and ubiquitin. it is wise to use at least two reference gene for one study may not be suitable for another. Total 370 participants were present in this study. RESULT: In our study, 114(30.8%) COVID positive patients were in E GENE GR 11-20, 147(39.7%) COVID positive patients were in E GENE GR 21-30 and 109(29.5%) COVID positive patients were in E GENE GR 31-40. 163(44.1%) COVID positive patients were in N GENE GR 11-20, 132(35.7%) COVID positive patients were in N GENE GR 21-30 and 75(20.3%) COVID positive patients were in N GENE GR 31-40. 228(61.6%) COVID positive patients were in RdRp GENE GR 11-20, 112(30.3%) COVID positive patients were in RdRp GENE GR 21-30 and 30(8.1%) COVID positive patients were in RdRp GENE GR 31-40. We found that, 30(8.1%) COVID positive patients died and 340 (91.9%) patients have survived. CONCLUSION: The patients who died had signicantly lower RT-PCR CT-values OF E. GENE, N.GENE, RdRp GENE than patients who survived. We concluded that the time of onset of symptoms were earlier for patients with lower CTvalue, than the other group who survived. So we concluded after correlating CT-values of real time RT-PCR and E. GENE, N.GENE, RdRp GENE, that the patients with lower CTvalues had more severity and had poor prognosis than the other group with higher RT-PCR CT-values of E.GENE, N, GENE, RdRp GENE

scholarly journals Development and application of a TaqMan-MGB real-time RT-PCR assay for the detection of porcine epidemic diarrhoea virus strains in China

2016 ◽  
Vol 60 (2) ◽  
pp. 127-133 ◽  
Author(s):  
Yi-Xuan Hou ◽  
Chun Xie ◽  
Kang Wang ◽  
Yu-Ting Zhao ◽  
Yang-Yang Xie ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
N Gene ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Conserved Sequence ◽  
Porcine Epidemic Diarrhoea Virus ◽  
Diarrhoea Virus ◽  
Quantitative Pcr Assay

AbstractIntroduction:A real-time RT-PCR method for identification and quantification of porcine epidemic diarrhoea virus (PEDV) strains in China was developed.Material and Methods:Based on the conserved sequence of the PEDV nucleocapsid (N) gene, a primer pair and probe were designed to establish a TaqMan-MGB real-time RT-PCR assay for quantitative detection of the virus. The sequence was cloned into the pMD18-T vector and a series of diluted recombinant plasmids were used to generate a standard curve with an R2 value of 0.999.Results:The developed quantitative PCR assay detected viral titres as low as 0.1 TCID50with high specificity and no cross-reaction with other porcine viruses (PoRV, TGEV, PRRSV, or CSFV). The intra-batch and inter-batch coefficients of variation were both less than 1%, which indicated good reproducibility. Thirty clinical diarrhoea samples obtained from pigs in Shanghai and Fujian were analysed using this quantitative PCR assay. Out of these samples, 93.3% were found to be PEDV positive.Conclusion:This approach is suitable for clinical sample identification and pathogenesis studies.

scholarly journals Comparison of SARS-CoV2 N gene real-time RT-PCR targets and commercially available mastermixes

2020 ◽  
Author(s):  
Julianne R Brown ◽  
Denise O’Sullivan ◽  
Rui PA Pereira ◽  
Alexandra S Whale ◽  
Eloise Busby ◽  
...  
Keyword(s):  
Real Time ◽  
Lower Limit ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
N Gene ◽  
Rt Pcr ◽  
One Step ◽  
Improved Performance ◽  
Rna Transcript ◽  
Nose And Throat

ABSTRACTWe aim to test four one-step RT real-time mastermix options for use in SARS-CoV2 real-time PCR, with three primer/probe assays targeting the N gene. The lower limit of detection is determined using a SARS CoV2 N gene RNA transcript dilution series (to 1 copy/µl) and verified using 74 nose and throat swabs.The N2 assay demonstrates the most sensitive detection of SARS-Cov-2 RNA. Three of the four mastermixes performed well, with the Takara One Step PrimeScript™ III RT-PCR Kit mastermix demonstrating improved performance at the lower limit of detection.

scholarly journals Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

2004 ◽  
Vol 50 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Leo L M Poon ◽  
Kwok Hung Chan ◽  
On Kei Wong ◽  
Timothy K W Cheung ◽  
Iris Ng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Stool Samples ◽  
Pcr Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

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