scholarly journals Validation of Real-Time RT-PCR for Detection of SARS-CoV-2 in the Early Stages of the COVID-19 Outbreak Without Emergency Use Authorization Reagents in the Republic of Korea

2021 ◽  
Author(s):  
Yoon-Seok Chung ◽  
Nam-Joo Lee ◽  
Sang Hee Woo ◽  
Jeong-Min Kim ◽  
Heui Man Kim ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time ◽  
Target Genes ◽  
Limit Of Detection ◽  
Republic Of Korea ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real ◽  
Genomic Markers ◽  
The Republic

Abstract Background: A real-time reverse transcription polymerase chain reaction (RT-PCR) assay for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed to rapidly diagnose coronavirus disease 2019 (COVID-19). Early diagnosis of COVID-19 enables timely treatment and the implementation of public health measures. We validated the sensitivity, specificity, precision, linearity, accuracy, and robustness of a real-time RT-PCR-based assay for SARS-CoV-2 detection and compared the sensitivity and specificity of Emergency Use Authorization (EUA)-approved reagents for COVID-19 with those of the real-time RT-PCR method.Methods: The real-time RT-PCR-based assay was performed to specifically amplify genomic markers of SARS-CoV-2.Results: The real-time RT-PCR assay was highly specific for SARS-CoV-2, and did not amplify genomic fragments of 13 other viruses that cause respiratory diseases. The assay showed high linearity when conducted with a viral isolate from a patient with COVID-19 together with plasmids containing the target genes. The assay showed good repeatability and reproducibility, with a coefficient of variation of 3%, and detected SARS-CoV-2 with a limit of detection of 1 PFU/mL.Conclusions: The present real-time RT-PCR-based assay can diagnose COVID-19 with high accuracy and sensitivity. This approach is highly effective and can facilitate the early diagnosis of COVID-19 without the use of EUA reagents in the Republic of Korea.

scholarly journals Validation of real-time RT-PCR for detection of SARS-CoV-2 in the early stages of the COVID-19 outbreak in the Republic of Korea

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yoon-Seok Chung ◽  
Nam-Joo Lee ◽  
Sang Hee Woo ◽  
Jeong-Min Kim ◽  
Heui Man Kim ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time ◽  
Limit Of Detection ◽  
Qpcr Assay ◽  
Republic Of Korea ◽  
Early Stages ◽  
Good Repeatability ◽  
Repeatability And Reproducibility ◽  
The Republic ◽  
Sensitivity Specificity

AbstractA real-time reverse transcription polymerase chain reaction (RT-qPCR) assay that does not require Emergency Use Authorization (EUA) reagents was tested and validated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the early stages of the outbreak of coronavirus disease 2019 (COVID-19) in the Republic of Korea. Early diagnosis of COVID-19 enables timely treatment and the implementation of public health measures. We validated the sensitivity, specificity, precision, linearity, accuracy, and robustness of the RT-qPCR assay for SARS-CoV-2 detection and compared its performance with that of several EUA-approved kits. Our RT-qPCR assay was highly specific for SARS-CoV-2 as demonstrated by not amplifying 13 other viruses that cause respiratory diseases. The assay showed high linearity using a viral isolate from a patient with known COVID-19 as well as plasmids containing target SARS-CoV-2 genes as templates. The assay showed good repeatability and reproducibility with a coefficient of variation of 3%, and a SARS-CoV-2 limit of detection of 1 PFU/mL. The RT-qPCR-based assay is highly effective and can facilitate the early diagnosis of COVID-19 without the use of EUA-approved kits or reagents in the Republic of Korea.

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S134-S135
Author(s):  
T Berent ◽  
T Rothstein ◽  
S Buckwalter ◽  
R Patel
Keyword(s):  
Real Time ◽  
Whole Blood ◽  
New Technologies ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Bartonella Species ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Hybridization Probes ◽  
Ffpe Samples

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

scholarly journals Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay

2008 ◽  
Vol 54 (2) ◽  
pp. 406-413 ◽  
Author(s):  
Weston C Hymas ◽  
Wade K Aldous ◽  
Edward W Taggart ◽  
Jeffery B Stevenson ◽  
David R Hillyard
Keyword(s):  
Sequence Analysis ◽  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Nucleotide ◽  
The Real ◽  
Nontranslated Region

Abstract Background: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5′ nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. Methods: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay–like plate end detection. Results: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. Conclusions: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.

scholarly journals Rapid SARS-CoV-2 antigen detection assay in comparison with real-time RT-PCR assay for laboratory diagnosis of COVID-19 in Thailand

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Chutikarn Chaimayo ◽  
Bualan Kaewnaphan ◽  
Nattaya Tanlieng ◽  
Niracha Athipanyasilp ◽  
Rujipas Sirijatuphat ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
False Negative ◽  
Laboratory Diagnosis ◽  
Antigen Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Screening Assay ◽  
The Real ◽  
Pcr Test

Abstract Background The Coronavirus disease 2019 (COVID-19) pandemic continues to spread across the world. Hence, there is an urgent need for rapid, simple, and accurate tests to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Performance characteristics of the rapid SARS-CoV-2 antigen detection test should be evaluated and compared with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19 cases. Methods The rapid SARS-CoV-2 antigen detection test, Standard™ Q COVID-19 Ag kit (SD Biosensor®, Republic of Korea), was compared with the real-time RT-PCR test, Allplex™ 2019-nCoV Assay (Seegene®, Korea) for detection of SARS-CoV-2 in respiratory specimens. Four hundred fifty-four respiratory samples (mainly nasopharyngeal and throat swabs) were obtained from COVID-19 suspected cases and contact individuals, including pre-operative patients at Siriraj Hospital, Bangkok, Thailand during March–May 2020. Results Of 454 respiratory samples, 60 (13.2%) were positive, and 394 (86.8%) were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. The duration from onset to laboratory test in COVID-19 suspected cases and contact individuals ranged from 0 to 14 days with a median of 3 days. The rapid SARS-CoV-2 antigen detection test’s sensitivity and specificity were 98.33% (95% CI, 91.06–99.96%) and 98.73% (95% CI, 97.06–99.59%), respectively. One false negative test result was from a sample with a high real-time RT-PCR cycle threshold (Ct), while five false positive test results were from specimens of pre-operative patients. Conclusions The rapid assay for SARS-CoV-2 antigen detection showed comparable sensitivity and specificity with the real-time RT-PCR assay. Thus, there is a potential use of this rapid and simple SARS-CoV-2 antigen detection test as a screening assay.

scholarly journals Primers and probe design and precision assessment of the real time RT-PCR assay in Coxsackievirus A10 and enterovirus detection

Data in Brief ◽  
2017 ◽  
Vol 12 ◽  
pp. 418-422
Author(s):  
Jingfang Chen ◽  
Rusheng Zhang ◽  
Xinhua Ou ◽  
Dong Yao ◽  
Zheng Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Probe Design ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real ◽  
Precision Assessment

scholarly journals Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay

2018 ◽  
Vol 2018 ◽  
pp. 1-7
Author(s):  
Lei Ma ◽  
Fanwen Zeng ◽  
Bihong Huang ◽  
Feng Cong ◽  
Ren Huang ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Limit Of Detection ◽  
Sybr Green ◽  
Watery Diarrhea ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Molecules ◽  
Positive Rate

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

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 Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay

2015 ◽  
Vol 53 (8) ◽  
pp. 2641-2647 ◽  
Author(s):  
Todd N. Wylie ◽  
Kristine M. Wylie ◽  
Richard S. Buller ◽  
Maria Cannella ◽  
Gregory A. Storch
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Limit Of Detection ◽  
Respiratory Illness ◽  
The United States ◽  
Human Enterovirus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcriptase Pcr ◽  
Enterovirus D68

We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.

scholarly journals Quantification of Substance P mRNA in Human Immune Cells by Real-Time Reverse Transcriptase PCR Assay

2002 ◽  
Vol 9 (1) ◽  
pp. 138-143 ◽  
Author(s):  
Jian-Ping Lai ◽  
Steven D. Douglas ◽  
Farida Shaheen ◽  
David E. Pleasure ◽  
Wen-Zhe Ho
Keyword(s):  
Substance P ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Immune Cells ◽  
Fetal Brain ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real ◽  
Human Immune

ABSTRACT We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.

scholarly journals Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

2004 ◽  
Vol 70 (7) ◽  
pp. 4371-4374 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real

ABSTRACT In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

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