scholarly journals Poor sensitivity of "AccuPower SARS-CoV-2 real time RT-PCR kit (Bioneer, South Korea)"

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Byron Freire-Paspuel ◽  
Miguel Angel Garcia-Bereguiain
Keyword(s):  
Developing Countries ◽  
South Korea ◽  
Real Time ◽  
Limit Of Detection ◽  
Negative Impact ◽  
Clinical Performance ◽  
Evaluation Studies ◽  
Government Agencies ◽  
Clinical Use ◽  
Rt Pcr

Abstract Background Several molecular kits are available for SARS-CoV-2 diagnosis, mostly lacking of proper clinical evaluation due to the emergency caused by COVID19 pandemia, particularly at developing countries like Ecuador. Objective We carried out an evaluation of the clinical performance of "AccuPower SARS-CoV-2 Real Time RT-PCR kit" (Bioneer, South Korea) for SARS-CoV-2 diagnosis using 2019-nCoV CDC EUA kit (IDT, USA) as a gold standard. Results 48 clinical specimens were included on the study, 38 tested SARS-CoV-2 positive and 10 SARS-CoV-2 negative for 2019-nCoV CDC EUA kit. For "AccuPower SARS-CoV-2 Real Time RT-PCR kit", only 30 were SARS-CoV-2 positive, indicating a low clinical performance with sensitivity of 78.9%. Moreover, the limit of detection for "AccuPower SARS-CoV-2 Real Time RT-PCR kit" was estimated to be higher than 40,000 viral RNA copies/mL of sample. Conclusions Proper clinical performance evaluation studies from government agencies at developing countries should be mandatory prior to clinical use authorization of SARS-CoV-2 diagnosis kits, particularly when those kits lack of either FDA or its country of origin clinical use authorization, to prevent the distribution of low quality products that may have a negative impact of COVID19 surveillance at developing countries.

scholarly journals Analytical and Clinical Evaluation of Two RT-qPCR SARS-CoV-2 Diagnostic Tests with Emergency Use Authorization in Ecuador

2021 ◽  
Author(s):  
Byron Freire-Paspuel ◽  
Alfredo Bruno ◽  
Alberto Orlando ◽  
Miguel Angel Garcia-Bereguiain
Keyword(s):  
Developing Countries ◽  
Gold Standard ◽  
Diagnostic Tests ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Evaluation Studies ◽  
Country Of Origin ◽  
Analytical Sensitivity ◽  
Clinical Use ◽  
Deep Impact

Dozens of RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA) or at least by a responsible agency of their country of origin, but many of them lack proper evaluation studies because of COVID-19 pandemic emergency. We evaluated the clinical performance of two commercially available kits in South America, the 2019-nCoV kit (Da An Gene, Guangzhou, China) and GenomeCoV19 kit (ABM, Richmond, Canada), for RT-qPCR SARS-CoV-2 diagnosis using the FDA EUA 2019-nCoV CDC kit (IDT, Coralville, IA) as gold standard. We found striking differences among clinical performance and analytical sensitivity in both kits; whereas the 2019-nCoV kit (Da An Gene) has a limit of detection of 2,000 copies/mL and 100% of sensitivity, the GenomeCoV19 kit (ABM) has a poor sensitivity of 75% and a limit of detection estimated to be over 8.000 copies/mL. The GenomeCoV19 kit (ABM) lacks clinical use authorization in Canada; however, the 2019-nCoV kit (Da An Gene) is authorized by the Chinese CDC. Our results support that only SARS-CoV-2 diagnosis kits with clinical use authorization from their country of origin should be exported to developing countries lacking proper evaluation agencies to avoid a deep impact of the COVID-19 pandemic due to unreliable diagnosis.

scholarly journals Analytical and Clinical Evaluation of “AccuPower SARS-CoV-2 Multiplex RT-PCR kit (Bioneer, South Korea)” and “Allplex 2019-nCoV Assay (Seegene, South Korea)” for SARS-CoV-2 RT-PCR Diagnosis: Korean CDC EUA as a Quality Control Proxy for Developing Countries

2021 ◽  
Vol 11 ◽  
Author(s):  
Byron Freire-Paspuel ◽  
Miguel Angel Garcia-Bereguiain
Keyword(s):  
Developing Countries ◽  
Quality Control ◽  
South Korea ◽  
Clinical Evaluation ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
High Sensitivity ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Diagnosis

BackgroundMultiple RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with Emergency Use Authorization (EUA) by FDA or their country of origin agency, but many of them lack of proper clinical evaluation.ObjectiveWe evaluated the clinical performance of two Korean SARS-CoV-2 RT-PCR kits available in South America, AccuPower SARS-CoV-2 Multiplex RT-PCR kit (Bioneer, South Korea) and Allplex 2019-nCoV Assay (Seegene, South Korea), for RT-qPCR SARS-CoV-2 diagnosis using the CDC protocol as a gold standard.ResultsWe found strong differences among both kits clinical performance and analytical sensitivity; while the Allplex 2019-nCoV Assay has sensitivity of 96.5% and an estimated limit of detection of 4,000 copies/ml, the AccuPower SARS-CoV-2 Multiplex RT-PCR kit has a sensitivity of 75.5% and limit of detection estimated to be bigger than 20,000 copies/ml.ConclusionsAccuPower SARS-CoV-2 Multiplex RT-PCR kit and Allplex 2019-nCoV Assay are both made in South Korea but EUA by Korean CDC was only granted to the later. Our results support that Korean CDC EUA should be considered as a quality control proxy for Korean SARS-CoV-2 RT-PCR kits prior to importation by developing countries to guarantee high sensitivity diagnosis.

scholarly journals Development and Evaluation of AccuPower® COVID-19 Multiplex Real-Time RT-PCR Kit and AccuPower® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit for SARS-CoV-2 Detection in Sputum, NPS/OPS, Saliva and Pooled Samples

2021 ◽  
Author(s):  
In Bum Suh ◽  
Jaegyun Lim ◽  
Hyo Seon Kim ◽  
Guil Rhim ◽  
Heebum Kim ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Rna Extraction ◽  
Kappa Coefficient ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Current Standard ◽  
Pooled Samples

Rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the successful control of the current global COVID-19 pandemic. The real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) is the most widely used detection technique. This research describes the development of two novel multiplex real-time RT-PCR kits, AccuPower ® COVID-19 Multiplex Real-Time RT-PCR Kit (NCVM) specifically designed for use with the ExiStation ™48 system (comprised of ExiPrep ™48 Dx and Exicycler ™96 by BIONEER, Korea) for sample RNA extraction and PCR detection, and AccuPower ® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit (SCVM) designed to be compatible with manufacturers` on-market PCR instruments. The limit of detection (LoD) of SCVM was 2 copies/µ L and the LoD of the NCVM was 120 copies/mL for both the gene and the SARS-CoV-2 gene (N gene and RdRp gene). The AccuPower ® kits demonstrated high precision with no cross reactivity to other respiratory-related microorganisms. The clinical performance of AccuPower ® kits was evaluated using the following clinical samples: sputum and nasopharyngeal/oropharyngeal swab (NPS/OPS) samples. Overall agreement of the AccuPower ® kits with a Food and Drug Administration (FDA) approved emergency use authorized commercial kit (STANDARD ™ M nCoV Real-Time Detection kit, SD BIOSENSOR, Korea) was above 95% (Cohen`s kappa coefficient ≥ 0.95), with a sensitivity of over 95%. The NPS/OPS specimen pooling experiment was conducted to verify the usability of AccuPower ® kits on pooled samples and the results showed greater than 90% agreement with individual NPS/OPS samples. The clinical performance of AccuPower ® kits with saliva samples was also compared with NPS/OPS samples and demonstrated over 95% agreement (Cohen`s kappa coefficient > 0.95). This study shows the BIONEER NCVM and SCVM assays are comparable with the current standard confirmation assay and are suitable for effective clinical management and control of SARS-CoV-2.

scholarly journals Design of a Novel Multiplex Real Time RT-PCR Assay for SARS-CoV-2 Detection

2020 ◽  
Author(s):  
Wei Zhen ◽  
Gregory J. Berry
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Cost Effective ◽  
Surface Glycoprotein ◽  
Taqman Probe ◽  
Epidemiological Surveillance ◽  
Plate Versus ◽  
Rt Pcr ◽  
Percent Agreement

AbstractCoronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 386,000 deaths globally as of June 4, 2020. In this study, we developed a novel multiplex real time reverse transcription (RT)-PCR test for detection of SARS-CoV-2, with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) of SARS-CoV-2 and a hydrolysis Taqman probe designed to specifically detect SARS-CoV-2. Following our design, we evaluated the Limit of detection (LOD) and clinical performance of this laboratory-developed test (LDT). A LOD study with inactivated whole virus exhibited equal performance to that seen in the modified CDC assay with a final LOD of 1,301 ± 13 genome equivalents/ml for our assay vs 1,249 ± 14 genome equivalents/ml for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal (NP) swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement with the modified CDC assay. The multiplex design of this assay allows the testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents during a time when both of these parameters have been critical. Our results demonstrate that our multiplex assay performs as well as the modified CDC assay, but is more efficient and cost effective and is therefore adequate for use as a diagnostic assay and for epidemiological surveillance and clinical management of SARS-CoV-2.

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 Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection

2020 ◽  
Vol 21 (16) ◽  
pp. 5674
Author(s):  
Cyril Chik-Yan Yip ◽  
Siddharth Sridhar ◽  
Kit-Hang Leung ◽  
Anthony Chin-Ki Ng ◽  
Kwok-Hung Chan ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Single Tube ◽  
Highly Sensitive ◽  
Testing Algorithms ◽  
Pcr Assays ◽  
Human Coronaviruses ◽  
Pooled Sample

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.

scholarly journals Validation of a Non-Specific Dye Real-Time PCR Assay for Porcine Adulteration in Meatball Using ND5 Primer

2017 ◽  
Vol 17 (2) ◽  
pp. 167 ◽  
Author(s):  
Tri Joko Raharjo ◽  
Ery Nourika Alfiraza ◽  
Esti Enjelina ◽  
Deni Pranowo
Keyword(s):  
Real Time ◽  
Annealing Temperature ◽  
Limit Of Detection ◽  
Melting Curve ◽  
High Specificity ◽  
Optimization Method ◽  
Amyl Alcohol ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Temperature Optimization

Porcine adulteration in meatball samples were analyzed using real-time polymerase chain reaction (RT-PCR), based on the ND5 primer obtained by previous study. This work consisted of three stages which were annealing temperature optimization, method validation, and application. DNA template was extracted using phenol-CIAA (chloroform-iso amyl alcohol) method. The optimum annealing temperature for ND5 primers (forward primer 5'-CATTCGCCTCACTCACATTAACC-3' and reverse primer 5'-AAGAGAGAGTTCTACGGTCTGTAG-3') was 58.0 °C, obtained after testing annealing at 50.5 to 59.5 °C gradient temperature with 5 °C interval. Melting curve analysis was done at 65.0 to 95.0 °C, with increasing temperature for 0.5 °C per 2 sec. Method was validated for its specificity, precision and limit of detection. RT-PCR method with ND5 primers produced 227 bp DNA fragment with 78.50 °C Tm value. From eight commercial meatball samples, one was detected containing porcine. The methods showed high specificity and precision, with experimentally determined limits for porcine were no less than 1%.

scholarly journals Foetal Wastage Among Ruminants Slaughtered at the Abattoir in Gwagwalada, FCT, Nigeria

2018 ◽  
Vol 1 (1) ◽  
pp. 53-64
Author(s):  
C. E Obudu
Keyword(s):  
Climate Change ◽  
Developing Countries ◽  
Negative Impact ◽  
Animal Protein ◽  
Government Agencies ◽  
Financial Loss ◽  
Protein Loss ◽  
Economic Implications ◽  
Potential Source ◽  
Comprehensive Examination

There is a chronic deficiency in the animal protein content of the diets of people in developing countries, including Nigeria. This has been attributed to low productivity of indigenous breeds of livestock, inadequate feed supplies, diseases and recently, climate change. However, foetal losses from slaughtered food animals have been identified as a potential source of animal protein loss but its overall negative impact on the national economy has not been evaluated. This study was therefore carried out to draw attention about the consequences of the practice and to proffer some recommendations. A five – month study was carried out to investigate the incidence of foetal wastage in ruminants slaughtered at the Gwagwalada abattoir in the Federal Capital Territory (FCT), Abuja, Nigeria as well as its economic implications. Results indicated that the incidence was 29.02%, 24.9% and 7.68% for cattle, sheep and goats respectively. Most of the wastage occurred in the first and second trimesters of pregnancy, while the total financial loss from the 260 foetuses recorded in the study was put at about N10.5 million. There is therefore the need for a more detailed and comprehensive examination of female animals prior to slaughter. Those diagnosed to be pregnant should be purchased and nurtured by relevant government agencies until delivery and the owners compensated. In addition, public enlightenment campaigns discouraging farmers from slaughtering obviously pregnant animals should be mounted, while violators should be made to pay fines..

scholarly journals Triplex Real Time RT-PCR for N1, N2 and RP probes from CDC EUA SARS-CoV-2 diagnosis kit

2020 ◽  
Author(s):  
Byron Freire-Paspuel ◽  
Patricio Vega-Mariño ◽  
Alberto Velez ◽  
Marilyn Cruz ◽  
Miguel Angel Garcia-Bereguiain
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Reverse Transcription ◽  
Clinical Performance ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Speed Up

AbstractCDC protocol for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include 3 targets for detection (N1, N2 and RP) labelled with FAM so 3 PCR reactions are required per sample. We developed a triplex, real-time reverse transcription PCR for SARS-CoV-2 that maintained clinical performance compared with CDC singleplex assay. This protocol could speed up detection and save reagents during current SARS-CoV-2 testing supplies shortage.

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