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 Rapid isothermal amplification and portable detection system for SARS-CoV-2

2020 ◽  
Vol 117 (37) ◽  
pp. 22727-22735 ◽  
Author(s):  
Anurup Ganguli ◽  
Ariana Mostafa ◽  
Jacob Berger ◽  
Mehmet Y. Aydin ◽  
Fu Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Alternative Pathway ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Complete Agreement ◽  
Rt Pcr ◽  
Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

scholarly journals A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

2020 ◽  
Vol 21 (8) ◽  
pp. 2826 ◽  
Author(s):  
Renfei Lu ◽  
Xiuming Wu ◽  
Zhenzhou Wan ◽  
Yingxue Li ◽  
Xia Jin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

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 Comparison of SARS-CoV2 N gene real-time RT-PCR targets and commercially available mastermixes

2020 ◽  
Author(s):  
Julianne R Brown ◽  
Denise O’Sullivan ◽  
Rui PA Pereira ◽  
Alexandra S Whale ◽  
Eloise Busby ◽  
...  
Keyword(s):  
Real Time ◽  
Lower Limit ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
N Gene ◽  
Rt Pcr ◽  
One Step ◽  
Improved Performance ◽  
Rna Transcript ◽  
Nose And Throat

ABSTRACTWe aim to test four one-step RT real-time mastermix options for use in SARS-CoV2 real-time PCR, with three primer/probe assays targeting the N gene. The lower limit of detection is determined using a SARS CoV2 N gene RNA transcript dilution series (to 1 copy/µl) and verified using 74 nose and throat swabs.The N2 assay demonstrates the most sensitive detection of SARS-Cov-2 RNA. Three of the four mastermixes performed well, with the Takara One Step PrimeScript™ III RT-PCR Kit mastermix demonstrating improved performance at the lower limit of detection.

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 Comparison of real-time RT-PCR and RT-PCR/MALDI-TOF methods for SARS-CoV-2 detection in saliva

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S9-S9
Author(s):  
Matthew M Hernandez ◽  
Radhika Banu ◽  
Paras Shrestha ◽  
Armi Patel ◽  
Feng Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
N Gene ◽  
Rt Pcr ◽  
Upper Respiratory ◽  
High Level ◽  
Respiratory Specimens ◽  
Pcr Test ◽  
Roche Cobas

Abstract Background The coronavirus disease 2019 pandemic has accelerated the need for rapid validation and implementation of assays for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in diagnostic specimens. Multiple molecular methods have received emergency use authorization by the U.S. Food and Drug Administration for detection of SARS-CoV-2 in upper respiratory specimens, with testing of nasopharyngeal (NP) specimens serving as the foundation for these assays. However, supply chain constraints and the need for improved ease and safety of collection have prompted consideration of other specimen types as alternatives to NP specimens for detection of SARS-CoV-2. Here, we compared two methods for SARS-CoV-2 detection in saliva: the Roche cobas® 6800 SARS-CoV-2 real-time RT-PCR Test (“Roche”), which tests for viral ORF1ab (target 1, T1) and envelope E genes (target 2, T2); and the Agena Biosciences MassARRAY® SARS-CoV-2 Panel/MassARRAY® System (“Agena”), which tests for targets in the ORF1ab gene (ORF1, Orf1ab) and nucleocapsid N gene (N1, N2, N3). Methods Sixty saliva specimens collected within 48 hours of SARS-CoV-2 detection in an upper respiratory (anterior nares or NP) specimen from the same individual were tested in both the Roche and Agena platforms. Each system was evaluated for overall detection results and agreement with results of matched upper respiratory specimens. In addition, we determined the limit of detection (LoD) for each system and its component targets using an in-house SARS-CoV-2 standard generated from pooled positive saliva specimens quantitated against a commercially available standard (ZeptoMetrix NATSARS(COV2)-ERC). Results Both platforms demonstrated a similarly high sensitivity (97%) and specificity (100%) when compared to matched patient upper respiratory specimens and had high agreement with one another (Cohen’s κ = 0.9321, p = 2.6x10-13). Overall, the LoD (copies/mL) for the Roche assay was four times lower than that of Agena for saliva specimens (390.6 v. 1562.5). Furthermore, we determined that the LoD differed among the target components of each assay. The experimental LoD was comparable across Roche targets, but probit analyses indicate T2 has greater sensitivity (LoD: 228.6), Of the five Agena targets, the N2 target had the lowest LoD (1562.5). Conclusions In sum, we demonstrate that saliva is an acceptable specimen for testing in both the Roche cobas® 6800 SARS-CoV-2 real-time RT-PCR Test and the Agena Biosciences MassARRAY® SARS-CoV-2 Panel/MassARRAY® System, and both provide sensitive and specific detection of SARS-CoV-2 in saliva specimens. Although there was a high level of agreement between platforms, the LoD was lower for the Roche compared to the Agena assay with T2 and N2 being the most sensitive targets on each platform, respectively. The addition of saliva as an acceptable specimen and understanding the sensitivity for testing on these platforms can further inform public health measures for screening and detection to combat the pandemic.

scholarly journals Relative Sensitivity of Common Target Genes For The Detection of SARS-Cov-2 In Real Time-PCR

2021 ◽  
Author(s):  
Reza Valadan ◽  
Soheila Golchin ◽  
Reza Alizadeh-Navaei ◽  
Mohammadreza Haghshenas ◽  
Mehryar Zargari ◽  
...  
Keyword(s):  
Real Time ◽  
Target Genes ◽  
False Negative ◽  
Rna Extraction ◽  
Host Cells ◽  
N Gene ◽  
Clinical Samples ◽  
Human Beings ◽  
Gene Expressions ◽  
Mazandaran Province

Abstract SARS-CoV-2(COVID-19) currently is the main cause of the severe acute respiratory disease and fatal outcomes in human beings worldwide. Several genes are used as targets for the detection of SARS-CoV-2, including the RDRP, N, and E genes. The present study aimed to determine the RDRP, N, and E genes expressions of SARS-CoV- 2 in clinical samples. For this purpose, 100 SARS-CoV-2 positive samples were collected from diagnostic laboratories of Mazandaran province, Iran. After RNA extraction, the real time RT-PCR assay was performed for differential gene expressions’ analysis of N, E, and RDRP. The CT values for N, RDRP, and E targets of 100 clinical samples for identifying SARS-CoV-2 were then evaluated using qRT-PCR. This result suggests N gene as a potential target for the detection of the SARS‐CoV‐2, since it was observed to be highly expressed in the nasopharyngeal or oropharynges of COVID-19 patients (P < 0.0001). Herein, we showed that SARS-CoV- 2 genes were differentially expressed in the host cells. Therefore, to reduce obtaining false negative results and to increase the sensitivity of the available diagnostic tests, the target genes should be carefully selected based on the most expressed genes in the cells.

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.

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.

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