Novel coronavirus (2019-nCoV) real-time RT-PCR N gene 2020 (Wuhan-N; 2019-nCoV-related screening test) v2 (protocols.io.bb3piqmn)

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

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 of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens

2020 ◽  
Vol 21 (7) ◽  
pp. 2574 ◽  
Author(s):  
Cyril Chik-Yan Yip ◽  
Chi-Chun Ho ◽  
Jasper Fuk-Woo Chan ◽  
Kelvin Kai-Wang To ◽  
Helen Shuk-Ying Chan ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Respiratory Viruses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Coronavirus Infection ◽  
Polymerase Chain ◽  
Novel Coronavirus

The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID50/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.

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 Evaluation of a real-time RT-PCR panel for detection of SARS-CoV-2 in bat guano

2021 ◽  
pp. 104063872199033
Author(s):  
Eman Anis ◽  
Greg Turner ◽  
Julie C. Ellis ◽  
Andrew Di Salvo ◽  
Amanda Barnard ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Global Health ◽  
Real Time ◽  
Health Concern ◽  
Rt Pcr ◽  
Infected People ◽  
Natural Reservoir ◽  
Rehabilitation Facilities ◽  
Novel Coronavirus ◽  
Bat Guano

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which is an ongoing global health concern. The exact source of the virus has not been identified, but it is believed that this novel coronavirus originated in animals; bats in particular have been implicated as the primary reservoir of the virus. SARS-CoV-2 can also be transmitted from humans to other animals, including tigers, cats, and mink. Consequently, infected people who work directly with bats could transfer the virus to a wild North American bat, resulting in a new natural reservoir for the virus, and lead to new outbreaks of human disease. We evaluated a reverse-transcription real-time PCR panel for detection of SARS-CoV-2 in bat guano. We found the panel to be highly specific for SARS-CoV-2, and able to detect the virus in bat guano samples spiked with SARS-CoV-2 viral RNA. Our panel could be utilized by wildlife agencies to test bats in rehabilitation facilities prior to their release to the wild, minimizing the risk of spreading this virus to wild bat populations.

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