One-step RT-PCR Ins214EPE assay for Omicron (B.1.1.529) variant detection v1

2021 ◽  
Author(s):  
Nikita Yolshin ◽  
Kirill Varchenko ◽  
Kseniya Komissarova ◽  
Daria Danilenko ◽  
Andrey Komissarov ◽  
...  
Keyword(s):  
N Gene ◽  
Clinical Samples ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Screening ◽  
Increased Risk ◽  
New Variant ◽  
Gene Assay ◽  
Research Institute

On 26 November 2021 WHO designated a new variant of concern B.1.1.529 named Omicron. This variant has a large number of mutations, some of which are concerning. Preliminary evidence suggests an increased risk of reinfection with this variant and reduced neutralization by convalescent and vaccinated sera, as compared to other VOCs. Implementation of the high-throughput rRT-PCR screening for Omicron is of great importance for monitoring the spread of this VOC in the population, especially in resource-limited countries lacking sufficient sequencing capacity. Omicron lineage B.1.1.529 (BA.1) has some indels that turned out to be a good target for its detection. In the current protocol, we use ins214EPE for this purpose. Here we describe the 1-step quantitative multiplex RT-qPCR assay consisting of the newly developed Ins214EPE detection set and widely used Hong Kong University N gene assay for SARS-CoV-2 detection (Chu et al., 2020). The assay was validated on the Omicron variant RNA kindly provided by the Pathogenic Microorganisms Variability Laboratory (Dr. Vladimir Guschin, Gamaleya Institute, Moscow, Russia) and RNA from the collection of Smorodintsev Research Institute of Influenza. Omicron RNA specimens were positive in the assay as expected. Negative controls were found negative. 10-fold serial dilutions of Omicron RNA were used to assess ins214EPE assay amplification efficiency. The amplification efficiency was 98,9% (R2 = 0,99). The developed rRT-PCR assay demonstrates high specificity. It was tested on 26 clinical samples (RNA extracted from oropharyngeal swabs) with previously characterized viruses belonging to 8 different SARS-CoV-2 lineages (including Delta B.1.617.2+AY.*) Specific signal was detected only in samples with SARS-CoV-2 Omicron lineage RNA (confirmed by whole-genome sequencing). Specificity was additionally tested on clinical samples positive for other respiratory viruses from the collection of Smorodintsev Research Institute of Influenza - influenza, parainfluenza, human seasonal coronaviruses (OC43, NL63, 229E, HKU1), hRSV, rhinoviruses, bocaviruses, metapneumovirus (33 in total) - with no false-positive results. Ins214EPE Cq 6x B.1.1.7 2x B.1.351 5x AT.1 6x B.1.617.2 4x AY.122 P.1 B.1.1.529 28,72 B.1.1.529 26,29 virus RP Cq SARS Cq Ins214 Cq RSV A 28,76 RSV A 30,56 RSV A 27,70 RSV B 31,49 RSV B 30,98 RSV B 32,33 NL63 32,20 NL63 30,42 NL63 24,95 Oc43 30,34 Oc43 30,69 Oc43 28,64 HKU1 30,06 HKU1 28,30 HKU1 30,73 229E 29,11 229E 32,52 229E 29,37 BoV 32,26 BoV 30,75 BoV 27,25 Rv 32,85 Rv 33,76 Rv 27,75 Piv1 28,63 Piv2 24,72 Piv3 27,01 Piv4 23,90 Adv 29,47 MPV 30,12 HIV A 29,13 HIV A 28,45 HIV A 28,16 39,06 c+ 34,15 26,61 28,44 Analytical sensitivity determination is underway. We consider developed assay to be useful in wide populational RT-PCR screening to assess the spread of Omicron variant.

scholarly journals Development and application of a method to detect 27 respiratory pathogens using multiplex RT-PCR combined with MassARRAY technology

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Huan Zhao ◽  
Yichao Yang ◽  
Jiangfeng Lyu ◽  
Xuyi Ren ◽  
Wei Cheng
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Early Recognition ◽  
Detection System ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Tract Infections

Abstract Background Respiratory tract infections are the most common infections that lead to morbidity and mortality worldwide. Early recognition and precise diagnosis of microbial etiology is important to treat LRTIs promptly, specifically and effectively. Objectives To establish a method based on multiplex reverse transcription (MRT)-PCR and MassARRAY technology for the simultaneous detection of 27 respiratory pathogens and explore its clinical application value. Methods Analytical sensitivity and specificity of the MRT-PCR-MassARRAY system were validated using inactivated bacterial and viral strains. Also we analyzed samples from 207 patients by MassARRAY methods and compared the results with consensus PCR/reverse transcription (RT)-PCR. Results The minimum detection limit of our MRT-PCR-MassARRAY method for pathogens was 10–100 copies/μl, with high specificity. Comparison test with consensus PCR/RT-PCR on 207 clinical samples, the positive, negative, and total correlation rates were 100, 98.68, and 99.03%, respectively. There was a high degree of agreement between the test results of the two methods (P < 0.01 by McNemar’s test). Conclusion Our detection system of 27 respiratory pathogens based on MassARRAY technology has high sensitivity and specificity, high throughput, and is simple to operate. It provides diagnostic value for the clinical diagnosis of respiratory pathogens and is of great significance in the screening of respiratory pathogens.

scholarly journals Identification of a polymorphism in the N gene of SARS-CoV-2 that adversely impacts detection by a widely-used RT-PCR assay

2020 ◽  
Author(s):  
Manu Vanaerschot ◽  
Sabrina A. Mann ◽  
James T. Webber ◽  
Jack Kamm ◽  
Sidney M. Bell ◽  
...  
Keyword(s):  
Diagnostic Sensitivity ◽  
N Gene ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Multiple Targets ◽  
Epidemiologic Evidence ◽  
Gene Assay ◽  
Pcr Primer

AbstractWe identify a mutation in the N gene of SARS-CoV-2 that adversely affects annealing of a commonly used RT-PCR primer; epidemiologic evidence suggests the virus retains pathogenicity and competence for spread. This reinforces the importance of using multiple targets, preferably in at least 2 genes, for robust SARS-CoV-2 detection.Article Summary LineA SARS-CoV-2 variant that occurs worldwide and has spread in California significantly affects diagnostic sensitivity of an N gene assay, highlighting the need to employ multiple viral targets for detection.

scholarly journals SARS-CoV-2 N Gene G29195T Point Mutation May Affect Diagnostic Reverse Transcription-PCR Detection

2022 ◽  
Author(s):  
Karrie K. K. Ko ◽  
Nurdyana Binte Abdul Rahman ◽  
Shireen Yan Ling Tan ◽  
Kenneth X. L. Chan ◽  
Sui Sin Goh ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Large Scale ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Viral Genomes ◽  
Reverse Transcription Pcr ◽  
Pcr Assays ◽  
Diagnostic Detection ◽  
Complementary Binding

Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples.

scholarly journals OmniSARS2: A Highly Sensitive and Specific RT-qPCR-Based COVID-19 Diagnostic Method Designed to Withstand SARS-CoV-2 Lineage Evolution

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1314
Author(s):  
Eduarda Carvalho-Correia ◽  
Carla Calçada ◽  
Fernando Branca ◽  
Nuria Estévez-Gómez ◽  
Loretta De Chiara ◽  
...  
Keyword(s):  
Cross Reactivity ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
S Gene ◽  
Genomic Knowledge ◽  
Gene Assay ◽  
One Step

Extensive transmission of SARS-CoV-2 during the COVID-19 pandemic allowed the generation of thousands of mutations within its genome. While several of these become rare, others largely increase in prevalence, potentially jeopardizing the sensitivity of PCR-based diagnostics. Taking advantage of SARS-CoV-2 genomic knowledge, we designed a one-step probe-based multiplex RT-qPCR (OmniSARS2) to simultaneously detect short fragments of the SARS-CoV-2 genome in ORF1ab, E gene and S gene. Comparative genomics of the most common SARS-CoV-2 lineages, other human betacoronavirus and alphacoronavirus, was the basis for this design, targeting both highly conserved regions across SARS-CoV-2 lineages and variable or absent in other Coronaviridae viruses. The highest analytical sensitivity of this method for SARS-CoV-2 detection was 94.2 copies/mL at 95% detection probability (~1 copy per total reaction volume) for the S gene assay, matching the most sensitive available methods. In vitro specificity tests, performed using reference strains, showed no cross-reactivity with other human coronavirus or common pathogens. The method was compared with commercially available methods and detected the virus in clinical samples encompassing different SARS-CoV-2 lineages, including B.1, B.1.1, B.1.177 or B.1.1.7 and rarer lineages. OmniSARS2 revealed a sensitive and specific viral detection method that is less likely to be affected by lineage evolution oligonucleotide–sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.

scholarly journals Development and Application of a Method to Detect 27 Respiratory Pathogens Using Multiplex RT-PCR Combined with MassARRAY Technology

2021 ◽  
Author(s):  
Huan Zhao ◽  
Yichao Yang ◽  
Jiangfeng Lyu ◽  
Xuyi Ren ◽  
Wei Cheng
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Early Recognition ◽  
Detection System ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Tract Infections

Abstract Background: Respiratory tract infections are the most common infections that lead to morbidity and mortality worldwide. Early recognition and precise diagnosis of microbial etiology is important to treat LRTIs promptly, specifically and effectively.Objectives: To establish a method based on multiplex reverse transcription (MRT)-PCR and MassARRAY technology for the simultaneous detection of 27 respiratory pathogens and explore its clinical application value.Methods: Analytical sensitivity and specificity of the MRT-PCR-MassARRAY system were validated using inactivated bacterial and viral strains. Also we analyzed samples from 207 patients by MassARRAY methods and compared the results with consensus PCR/reverse transcription (RT)-PCR.Results: The minimum detection limit of our MRT-PCR-MassARRAY method for pathogens was 10–100 copies/µl, with high specificity. Comparison test with consensus PCR/RT-PCR on 207 clinical samples, the positive, negative, and total correlation rates were 100%, 98.68%, and 99.03%, respectively. There was a high degree of agreement between the test results of the two methods (P < 0.01 by McNemar’s test).Conclusion: Our detection system of 27 respiratory pathogens based on MassARRAY technology has high sensitivity and specificity, high throughput, and is simple to operate. It provides diagnostic value for the clinical diagnosis of respiratory pathogens and is of great significance in the screening of respiratory pathogens.

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.

scholarly journals Development of TaqMan-based real-time RT-PCR assay based on N gene for the quantitative detection of feline morbillivirus

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Siti Tasnim Makhtar ◽  
Sheau Wei Tan ◽  
Nur Amalina Nasruddin ◽  
Nor Azlina Abdul Aziz ◽  
Abdul Rahman Omar ◽  
...  
Keyword(s):  
Real Time ◽  
Measles Virus ◽  
Clinical Sample ◽  
N Gene ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
High Morbidity ◽  
Canine Distemper ◽  
Rt Pcr ◽  
Pcr Assay

Abstract Background Morbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection. Results A specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34–0.53% and 1.38–2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR. Conclusions The TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.

scholarly journals Novel indel mutation in the N gene of SARS-CoV-2 clinical samples that were diagnosed positive in a commercial RT-PCR assay

2021 ◽  
pp. 198398
Author(s):  
Sanghoo Lee ◽  
DongJu Won ◽  
Chang-Ki Kim ◽  
Jinwoo Ahn ◽  
Youngkee Lee ◽  
...  
Keyword(s):  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Indel Mutation

scholarly journals The role of sub-genomic RNA in discordant results from RT-PCR tests for COVID-19.

2021 ◽  
Author(s):  
Noah Toppings ◽  
Lisa Oberding ◽  
Yi-Chan Lin ◽  
David Evans ◽  
Dylan R Pillai
Keyword(s):  
Gene Copy ◽  
N Gene ◽  
Clinical Samples ◽  
Genomic Rna ◽  
Rt Pcr ◽  
E Gene ◽  
Reverse Transcription Pcr ◽  
Inconclusive Result ◽  
Infected Cells ◽  
Eclipse Phase

Reverse transcription-PCR (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when one RT-PCR target is positive for SARS-CoV-2 and one RT-PCR target is negative within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. It was hypothesized that concentration differences across targets may be due to the transcription of sub-genomic RNA, as this would result in an increase in the concentration of gene targets near the 3’ end of the SARS-CoV-2 genome.  A panel of six digital droplet (dd)PCR assays was designed to quantitate the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity in such a way as to maximize gene-to-gene copy ratios. Eleven clinical nasopharyngeal swabs were also tested with this panel. In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. Both the highest specific infectivity (copies/pfu) and the highest differences between gene targets were observed at 6 hours post-infection (eclipse phase) in infected cells. The same trends in the relative abundance of copies across different targets observed in infected cells was observed in clinical samples, though trends were more pronounced in infected cells. This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by this data.

scholarly journals Development of Taqman-Based Real-Time RT-PCR Assay Based on N Gene for The Quantitative Detection of Feline Morbillivirus

2020 ◽  
Author(s):  
Siti Tasnim Makhtar ◽  
Sheau Wei Tan ◽  
Nur Amalina Nasruddin ◽  
Nor Azlina Abdul Aziz ◽  
Abdul Rahman Omar ◽  
...  
Keyword(s):  
Real Time ◽  
Measles Virus ◽  
Clinical Sample ◽  
Feline Leukemia Virus ◽  
N Gene ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Canine Distemper ◽  
Rt Pcr ◽  
Pcr Assay

Abstract Background: Feline morbillivirus (FeMV) is a member of genus Morbillivirus which has been associated with the chronic kidney disease in cats even though a definite relationship is still unclear. Morbilliviruses are associated with severe diseases such as Peste des petits ruminants, canine distemper and measles. FeMV has been detected in many countries including Malaysia. This study aims to develop a Taqman real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.Results: A specific assay was developed since no amplification was observed in viral strains from the same Paramyxoviridae family, such as canine distemper virus (CDV), Newcastle disease virus (NDV) and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 x 104 copies/L with Cq value of 34.32 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34% - 0.53% and 1.38% - 2.03%, respectively. Besides that, the clinical sample evaluation using this assay showed a higher detection rate, with 26 (37%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.Conclusions: The Taqman-based real-time RT PCR assay targeting the N gene described here is more sensitive, specific, rapid and reproducible compared to the conventional RT-PCR assay targeting the N gene and it could be used to detect early infection in cats.

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