Laboratory diagnosis challenges of Covid-19: a Real-time RT-PCR assay-based survey

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.

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Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638721994810 ◽

2021 ◽

pp. 104063872199481

Author(s):

Yixin Xiao ◽

Fan Yang ◽

Fumin Liu ◽

Hangping Yao ◽

Nanping Wu ◽

...

Keyword(s):

Avian Influenza ◽

Real Time ◽

Influenza A ◽

Minor Groove Binder ◽

Rt Pcr ◽

Influenza A Viruses ◽

Pcr Assay ◽

Infectious Dose ◽

The Matrix ◽

Taqman Minor Groove Binder

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.

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A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

Journal of Virological Methods ◽

10.1016/j.jviromet.2013.02.004 ◽

2013 ◽

Vol 189 (2) ◽

pp. 277-282 ◽

Cited By ~ 11

Author(s):

Yong Yan ◽

Heng-hui Wang ◽

Lei Gao ◽

Ji-mei Ji ◽

Zhi-jie Ge ◽

...

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Rt Pcr ◽

Pcr Assay ◽

Human Norovirus ◽

One Step

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Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

Journal of Clinical Virology ◽

10.1016/j.jcv.2009.09.033 ◽

2010 ◽

Vol 47 (1) ◽

pp. 54-59 ◽

Cited By ~ 16

Author(s):

Sylvie Pillet ◽

Geneviève Billaud ◽

Shabir Omar ◽

Bruno Lina ◽

Bruno Pozzetto ◽

...

Keyword(s):

Real Time ◽

R Gene ◽

Rt Pcr ◽

Pcr Assay ◽

Clinical Specimens

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Performance characteristics of a real-time RT-PCR assay for quantification of hepatitis C virus RNA in patients with genotype 1 and 2 infections

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2008.082 ◽

2008 ◽

Vol 46 (4) ◽

Cited By ~ 2

Author(s):

Jee-Fu Huang ◽

Chia-Yen Dai ◽

Ya-Yun Lin ◽

Ming-Lung Yu ◽

Shu-Fen Liu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Real Time ◽

Performance Characteristics ◽

Genotype 1 ◽

Rt Pcr ◽

Hepatitis C Virus Rna ◽

Pcr Assay ◽

Virus Rna

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Monitoring Current-Season Spread of Potato virus Y in Potato Fields Using ELISA and Real-Time RT-PCR

Plant Disease ◽

10.1094/pdis-03-12-0283-re ◽

2013 ◽

Vol 97 (5) ◽

pp. 641-644 ◽

Cited By ~ 17

Author(s):

Manphool S. Fageria ◽

Mathuresh Singh ◽

Upeksha Nanayakkara ◽

Yvan Pelletier ◽

Xianzhou Nie ◽

...

Keyword(s):

Real Time ◽

Potato Virus ◽

Potato Virus Y ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Pcr Assay ◽

Current Season ◽

Seed Market ◽

Polymerase Chain ◽

Time Of Harvest

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

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Improved detection of Sugarcane yellow leaf virus using a real-time fluorescent (TaqMan) RT-PCR assay

Journal of Virological Methods ◽

10.1016/s0166-0934(01)00406-2 ◽

2002 ◽

Vol 103 (2) ◽

pp. 109-120 ◽

Cited By ~ 75

Author(s):

J Korimbocus ◽

D Coates ◽

I Barker ◽

N Boonham

Keyword(s):

Real Time ◽

Yellow Leaf ◽

Rt Pcr ◽

Pcr Assay ◽

Sugarcane Yellow Leaf Virus ◽

Leaf Virus

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

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab191.287 ◽

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.

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