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

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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Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

10.21203/rs.3.rs-28883/v1 ◽

2020 ◽

Cited By ~ 8

Author(s):

Karina Helena Morais Cardozo ◽

Adriana Lebkuchen ◽

Guilherme Goncalves Okai ◽

Rodrigo Andrade Schuch ◽

Luciana Godoy Viana ◽

...

Keyword(s):

Real Time ◽

Large Scale ◽

Low Cost ◽

Large Population ◽

Virus Detection ◽

Standard Test ◽

Clinical Samples ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

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Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

10.21203/rs.3.rs-28883/v2 ◽

2020 ◽

Cited By ~ 1

Author(s):

Karina Helena Morais Cardozo ◽

Adriana Lebkuchen ◽

Guilherme Goncalves Okai ◽

Rodrigo Andrade Schuch ◽

Luciana Godoy Viana ◽

...

Keyword(s):

Real Time ◽

Large Scale ◽

Low Cost ◽

Large Population ◽

Virus Detection ◽

Standard Test ◽

Clinical Samples ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

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Reverse transcription-PCR using a Primer Set Targeting the 3D Region Detects Foot-and-Mouth Disease Virus with High Sensitivity

10.1101/305086 ◽

2018 ◽

Author(s):

Tatsuya Nishi ◽

Toru Kanno ◽

Nobuaki Shimada ◽

Kazuki Morioka ◽

Makoto Yamakawa ◽

...

Keyword(s):

Reverse Transcription ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Clinical Samples ◽

Rt Pcr ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Pcr Assays

AbstractBecause foot-and-mouth disease (FMD) has the potential to spread extensively, methods used for its diagnosis must be rapid and accurate. Therefore, reverse transcription-PCR (RT-PCR) plays an important diagnostic role. Here we designed the primer set FM8/9 to amplify 644 bases of the conserved 3D region of all seven serotypes of FMD virus (FMDV). We compared the performance of RT-PCR assays using FM8/9 with that using the primer set 1F/R targeting the 5’-UTR described in the manual of the World Organization for Animal Health. The detection limits of the RT-PCR assays were determined for 14 strains representing all serotypes. Compared with the sensitivities of the RT-PCR assay using 1F/R, those using FM8/9 were 101-to 104-fold higher for eight strains. To assess the validity of the methods for analyzing clinical samples, sera and saliva samples from pigs and cows infected with FMDV were collected daily and analyzed using the two PCR assays. The FM8/9 assay detected FMDV from all infected pigs and cows for longer times compared with the 1F/R assay, therefore revealing higher sensitivity for the clinical samples. Our results suggest that the FM8/9 RT-PCR assay is highly sensitive and is therefore suitable for the diagnosis of FMD.

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Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

Applied and Environmental Microbiology ◽

10.1128/aem.67.12.5593-5600.2001 ◽

2001 ◽

Vol 67 (12) ◽

pp. 5593-5600 ◽

Cited By ~ 64

Author(s):

Julie Jean ◽

Burton Blais ◽

André Darveau ◽

Ismaı̈l Fliss

Keyword(s):

Nucleic Acid ◽

Reverse Transcription ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Nucleic Acid Sequence ◽

Clinical Samples ◽

Rt Pcr ◽

Dot Blot ◽

Reverse Transcription Pcr ◽

Dot Blot Assay

ABSTRACT A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

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

10.1101/2021.12.14.21267750 ◽

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.

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Identification of Hotspot Mutations in the N Gene of SARS-CoV-2 in Russian Clinical Samples That May Affect the Detection by Reverse Transcription-PCR

Diagnostics ◽

10.3390/diagnostics12010147 ◽

2022 ◽

Vol 12 (1) ◽

pp. 147

Author(s):

Sergei A. Kiryanov ◽

Tatiana A. Levina ◽

Maria V. Konopleva ◽

Anatoly P. Suslov

Keyword(s):

Genetic Variability ◽

Variable Region ◽

N Gene ◽

Clinical Samples ◽

Rt Pcr ◽

E Gene ◽

Reverse Transcription Pcr ◽

Affect Detection ◽

Common Target

Sensitive and reliable diagnostic test systems based on real-time PCR are of great importance in the fight against the ongoing SARS-CoV-2 pandemic. The genetic variability of the SARS-CoV-2 virus leads to the accumulation of mutations, some of which may affect the sensitivity of modern PCR assays. The aim of this study was to search in Russian clinical samples for new mutations in SARS-CoV-2 gene N that can affect the detection by RT-PCR. In this study, the polymorphisms in the regions of the target gene N causing failed or poor detection of the target N in the RT-PCR assay on 12 selected samples were detected. Sequencing the entire N and E genes in these samples along with other 195 samples that were positive for both target regions was performed. Here, we identified a number of nonsynonymous mutations and one novel deletion in the N gene that affected the ability to detect a target in the N gene as well a few mutations in the E gene of SARS-CoV-2 that did not affect detection. Sequencing revealed that majority of the mutations in the N gene were located in the variable region between positions 193 and 235 aa, inside and nearby the phosphorylated serine-rich region of the protein N. This study highlights the importance of the further characterization of the genetic variability and evolution of gene N, the most common target for detecting SARS-CoV-2. The use of at least two targets for detecting SARS-CoV-2, including one for the E gene, will be necessary for reliable diagnostics.

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Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

10.1101/2020.03.02.20030130 ◽

2020 ◽

Cited By ~ 29

Author(s):

Weihua Yang ◽

Xiaofei Dang ◽

Qingxi Wang ◽

Mingjie Xu ◽

Qianqian Zhao ◽

...

Keyword(s):

Nucleic Acid ◽

Reverse Transcription ◽

Virus Disease ◽

Lamp Assay ◽

Nucleic Acid Amplification ◽

N Gene ◽

Nucleic Acid Detection ◽

Lamp Method ◽

Rt Pcr ◽

Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

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High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening

10.1101/2020.05.14.097295 ◽

2020 ◽

Cited By ~ 2

Author(s):

Rania Francis ◽

Marion Le Bideau ◽

Priscilla Jardot ◽

Clio Grimaldier ◽

Didier Raoult ◽

...

Keyword(s):

High Speed ◽

Large Scale ◽

Vaccine Development ◽

Work Load ◽

Drug Susceptibility Testing ◽

High Content Screening ◽

Clinical Samples ◽

Rt Pcr ◽

Positive Elements

AbstractSARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.

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Verification and diagnostic evaluation of the RealStar® Middle East respiratory syndrome coronavirus (N gene) reverse transcription-PCR kit 1.0

Future Microbiology ◽

10.2217/fmb-2019-0067 ◽

2019 ◽

Vol 14 (11) ◽

pp. 941-948 ◽

Cited By ~ 5

Author(s):

Leonie-Sophie Hecht ◽

Angeles Jurado-Jimenez ◽

Markus Hess ◽

Hussein El Halas ◽

Gregor Bochenek ◽

...

Keyword(s):

Middle East ◽

Reverse Transcription ◽

Diagnostic Procedure ◽

Middle East Respiratory Syndrome ◽

Diagnostic Evaluation ◽

N Gene ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Confirmatory Assay ◽

Pcr Targeting

Aim: We report the diagnostic evaluation of a confirmatory reverse transcription-PCR (RT-PCR) kit targeting the Middle East respiratory syndrome coronavirus (MERS-CoV) N gene. Material & methods: 33 patient samples from two collections sites in Riyadh, Saudi Arabia, which were pre-characterized via real-time RT-PCR targeting MERS-CoV orf1a and upE, and were tested using the MERS-CoV N gene, as a confirmatory assay. This diagnostic procedure follows a two-step diagnostics scheme, recommended by the WHO. Results: 18/33 samples tested positive, 11/33 tested negative for MERS-CoV RNA and 2/33 showed uncertain results. Conclusion: The results suggest, that the RealStar® MERS-CoV (N gene) RT-PCR kit 1.0 can be considered a suitable and reliable confirmatory assay in combination with the RealStar MERS-CoV RT-PCR kit 1.0 according to the diagnostic scheme recommended by WHO.

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