scholarly journals Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260487
Author(s):  
Justin S. Lee ◽  
Jason M. Goldstein ◽  
Jonathan L. Moon ◽  
Owen Herzegh ◽  
Dennis A. Bagarozzi ◽  
...  
Keyword(s):  
Real Time ◽  
False Positive ◽  
Bulk Material ◽  
Virus Detection ◽  
Rt Pcr ◽  
Nucleocapsid Gene ◽  
Positive Reactivity ◽  
Control And Prevention ◽  
Diagnostic Panel ◽  
Novel Coronavirus

At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the “bulk” manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3’ end of the N3 probe and the 3’ end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the “bulk” material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.

Establishment and evaluation of real-time PCR for West Nile virus detection

2009 ◽  
Vol 6 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Shi Li-Jun ◽  
Lu Mao-Min ◽  
Li Gang ◽  
Li Cheng-Yao ◽  
Zhang Jin-Gang
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Virus Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
West Nile ◽  
Capsid Protein Gene ◽  
Specific Test ◽  
Intercalating Dye ◽  
Polymerase Chain

AbstractA rapid real-time polymerase chain reaction (RT-PCR) for detecting West Nile virus (WNV) was established. Primers were designed according to the sequence of the capsid protein gene of WNV by Primer Premier 5.0. In this way, an inexpensive assay using the intercalating dye SYBR Green I was developed and validated. The amplifying curve showed that this method could successfully amplify 102 copies/μl of the WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay system showed high reproducibility with coefficient of variation (CV) <2%. Thus the newly established RT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV.

The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR

2010 ◽  
Vol 48 (2) ◽  
pp. 91-95 ◽  
Author(s):  
Chonticha Klungthong ◽  
Piyawan Chinnawirotpisan ◽  
Kittinun Hussem ◽  
Thipwipha Phonpakobsin ◽  
Wudtichai Manasatienkij ◽  
...  
Keyword(s):  
Real Time ◽  
Pandemic Influenza ◽  
Influenza A ◽  
Virus Detection ◽  
Rt Pcr ◽  
Influenza A H1n1 ◽  
The Impact

scholarly journals Review on Diagnostic Methods for SARS-CoV-2

Coronaviruses ◽  
2020 ◽  
Vol 01 ◽  
Author(s):  
Chandrashekhar Chauriya ◽  
Mahesh Sahu ◽  
Anuj Kumar Singh ◽  
Amita Verma
Keyword(s):  
Disease Control ◽  
Diagnostic Methods ◽  
World Health ◽  
Diagnostic Strategy ◽  
Serological Testing ◽  
Rt Pcr ◽  
Number Of Patients ◽  
Control And Prevention ◽  
Antigen Tests ◽  
Novel Coronavirus

Background: In December 2019, a large number of patients with a novel coronavirus were identified in Wuhan, China. The novel coronavirus (COVID-19) is highly contagious and increasing the rate of mortality day by day. The World Health Organization declared COVID-19 as a worldwide pandemic on March 11, 2020. Early diagnosis, of SARS-CoV-2, can restrict the COVID-19 pandemic. Objective: We aim to study the currently available diagnostic methods for COVID-19. Methods: World Health Organisation portal, Centre for Disease control and prevention portal, Indian Council of Medical Research portal, Chinese Centre for Disease Control and prevention portal, Science Direct, Google Scholar, Research Gate, etc. were searched for obtaining data. Results: Rapid diagnosis and prompt treatment can reduce the number of prospective cases. The diagnostic strategy encompasses the screening of virus with nucleic acid amplification test (NAAT) such as real-time reverse-transcription polymerase chain reaction (RT-PCR) assays. Serological testing is a diagnostic procedure used for identifying the presence of an immune responses. Radiological findings in individuals with COVID-19 were characterised by multiple areas of consolidation in chest. Rapid antigen tests are in- vitro diagnostics have been designed to give results within 10–20 min. Conclusion: Rapid, simple, and safe diagnosis of COVID-19 has a great impact on deciding clinical and epidemiological factors. RT-PCR results often require 5 to 6 hours. Diagnosis of by serological testing is not suitable but they are important epidemiologically. At present, the best radiological strategy remains undefined. Rapid antigen tests have limitations on sensitivity.

scholarly journals Pooling for SARS-CoV2 Surveillance: Validation and Strategy for Implementation in K-12 Schools

2021 ◽  
Vol 9 ◽  
Author(s):  
Alexandra M. Simas ◽  
Jimmy W. Crott ◽  
Chris Sedore ◽  
Augusta Rohrbach ◽  
Anthony P. Monaco ◽  
...  
Keyword(s):  
Financial Burden ◽  
Cost Savings ◽  
Educational Institutions ◽  
Repeated Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Nucleocapsid Gene ◽  
Specificity And Sensitivity ◽  
Novel Coronavirus ◽  
K 12

Repeated testing of a population is critical for limiting the spread of the SARS-CoV-2 virus and for the safe reopening of educational institutions such as kindergarten—grade 12 (K-12) schools and colleges. Many screening efforts utilize the CDC RT-PCR based assay which targets two regions of the novel Coronavirus nucleocapsid gene. The standard approach of testing each person individually, however, poses a financial burden to these institutions and is therefore a barrier to using testing for re-opening. Pooling samples from multiple individuals into a single test is an attractive alternate approach that promises significant cost savings—however the specificity and sensitivity of such approaches needs to be assessed prior to deployment. To this end, we conducted a pilot study to evaluate the feasibility of analyzing samples in pools of eight by the established RT-PCR assay. Participants (1,576) were recruited from amongst the Tufts University community undergoing regular screening. Each volunteer provided two swabs, one analyzed separately and the other in a pool of eight. Because the positivity rate was very low, we spiked approximately half of the pools with laboratory-generated swabs produced from known positive cases outside the Tufts testing program. The results of pooled tests had 100% correspondence with those of their respective individual tests. We conclude that pooling eight samples does not negatively impact the specificity or sensitivity of the RT-PCR assay and suggest that this approach can be utilized by institutions seeking to reduce surveillance costs.

scholarly journals Saving Resources: SARS-CoV-2 Diagnostics by Real-Time RT-PCR Using Reduced Reaction Volumes

Diseases ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 84
Author(s):  
Sabine Bock ◽  
Bernd Hoffmann ◽  
Martin Beer ◽  
Kerstin Wernike
Keyword(s):  
Real Time ◽  
Viral Genome ◽  
Virus Detection ◽  
World Health ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reaction Volume ◽  
Reaction Volumes ◽  
Health Organization ◽  
The Individual

Since the beginning of 2020, the betacoronavirus SARS-CoV-2 is causing a global pandemic of an acute respiratory disease termed COVID-19. The diagnostics of the novel disease is primarily based on direct virus detection by RT-PCR; however, the availability of test kits may become a major bottleneck, when millions of tests are performed per week. To increase the flexibility of SARS-CoV-2 diagnostics, three real-time RT-PCR assays listed on the homepage of the World Health Organization were selected and investigated regarding their compatibility with three different RT-PCR kits. Furthermore, the reaction volume of the PCR chemistry was reduced up to half of the original protocol to make the individual reactions more cost- and resource-effective. When testing dilution series of culture-grown virus, nearly identical quantification cycle values (Cq) were obtained for all RT-PCR assay/chemistry combinations. Regarding the SARS-CoV-2 detection in clinical samples, agreeing results were obtained for all combinations for virus negative specimens and swabs containing high to medium viral genome loads. In cases of very low SARS-CoV-2 genome loads (Cq > 36), inconsistent results were observed, with some test runs scoring negative and some positive. However, no preference of a specific target within the viral genome (E, RdRp, or N) or of a certain chemistry was seen. In summary, a reduction of the reaction volume and the type of PCR chemistry did not influence the PCR sensitivity.

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.

scholarly journals Detection and discrimination of influenza B Victoria lineage deletion variant viruses by real-time RT-PCR

2020 ◽  
Vol 25 (41) ◽  
Author(s):  
Bo Shu ◽  
Marie K Kirby ◽  
Christine Warnes ◽  
Wendy M Sessions ◽  
William G Davis ◽  
...  
Keyword(s):  
Real Time ◽  
Cross Reactivity ◽  
Influenza B ◽  
Diagnostic Assay ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Human Influenza Virus ◽  
Vaccine Recommendations ◽  
Geographical Regions ◽  
Diagnostic Panel

Background During the 2016/17 influenza season, influenza B/VIC lineage variant viruses emerged with two (K162N163) or three (K162N163D164) amino acid (aa) deletions in the haemagglutinin (HA) protein. There are currently five antigenically distinct HA proteins expressed by co-circulating influenza B viruses: B/YAM, B/VIC V1A (no deletion), B/VIC V1A-2DEL (2 aa deletion) and two antigenically distinguishable groups of B/VIC V1A-3DEL (3 aa deletion). The prevalence of these viruses differs across geographical regions, making it critical to have a sensitive, rapid diagnostic assay that detects and distinguishes these influenza B variant viruses during surveillance. Aim Our objective was to develop a real-time RT-PCR (rRT-PCR) assay for detection and discrimination of influenza B/VIC lineage variant viruses. Methods We designed a diagnostic assay with one pair of conserved primers and three probes specific to each genetic group. We used propagated influenza B/VIC variant viruses and clinical specimens to assess assay performance. Results This rRT-PCR assay detects and distinguishes the influenza B/VIC V1A, B/VIC V1A-2DEL, and B/VIC V1A-3DEL variant viruses, with no cross-reactivity. This assay can be run as a multiplex reaction, allowing for increased testing efficiency and reduced cost. Conclusion Coupling this assay with the Centers for Disease Control and Prevention’s Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza B Lineage Genotyping Kit results in rapid detection and characterisation of circulating influenza B viruses. Detailed surveillance information on these distinct influenza B variant viruses will provide insight into their prevalence and geographical distribution and could aid in vaccine recommendations.

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

2020 ◽  
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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