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

Yip, Cyril Chik-Yan; Sridhar, Siddharth; Leung, Kenneth; Ng, Anthony Chin-Ki; Chan, Kwok-Hung; Chan, Jasper Fuk‐Woo; Tsang, Owen Tak‐Yin; Hung, Ivan; Cheng, Vincent Chi-Chung; Yuen, Kwok Yung; To, Kelvin Kai‐Wang

doi:10.3390/ijms21165674

Cited by 25 publications

(26 citation statements)

References 35 publications

(47 reference statements)

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“…The male: female ratio was 136:160 and patients’ median age was 59.5 years (range: 3 months – 99 years). Some of these specimens were previously used in other assay evaluations published by our group [ 14 , 15 , 20 ]. In addition to clinical specimens, three proficiency testing (PT) samples from College of American Pathologist (CAP) and eight PT samples from Quality Control for Molecular Diagnostics (QCMD) with different concentrations of SARS-CoV-2 or negative for SARS-CoV-2 were also evaluated.…”

Section: Methodsmentioning

confidence: 99%

“…The second assay is an in-house developed STN real-time RT-PCR assay targeting the SARS-CoV-2 N gene was performed using QuantiNova Probe RT-PCR Kit (QIAGEN, Hilden, Germany) on LightCycler 480 II Real-Time PCR System (Roche) previously described by us [ 15 ]. This assay format maximizes sensitivity by combining two PCR reactions within a single reaction vessel.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, the best sample types and optimal RT-PCR assay design for pooled testing are unknown. This study had two objectives: we first evaluated the performance of a novel dual-target commercial kit for COVID-19 diagnosis and compared its performance to the LightMix E-gene kit and our previously validated in-house single-tube nested (STN) COVID-19-N RT-PCR assay [ 14 , 15 ]. We then examined the sensitivity of pooled testing of paired nasopharyngeal swab (NPS) and deep throat saliva samples in a range of pool sizes using three RT-PCR assays.…”

Section: Introductionmentioning

confidence: 99%

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Comparative evaluation of a dual-target real-time RT-PCR assay for COVID-19 diagnosis and assessment of performance in pooled saliva and nasopharyngeal swab samples

Yip

Leung

et al. 2021

Expert Review of Molecular Diagnostics

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Objectives : Sensitive molecular diagnostic assays are essential for COVID-19 diagnosis. We evaluated the Hecin Scientific SARS-CoV-2 nucleic acid test kit, a dual-target real-time RT-PCR assay targeting the SARS-CoV-2 N and ORF1ab genes. Methods : The Hecin test kit’s diagnostic performance in detecting SARS-CoV-2 RNA was compared to the LightMix Modular SARS and Wuhan CoV E-gene kit (TIB Molbiol) and an in-house single-tube nested real-time RT-PCR using 296 clinical specimens, 11 proficiency testing samples, and 30 low-positive deep throat saliva and nasopharyngeal swab (NPS) samples pooled into negative samples in ratios of 1:5, 1:10, and 1:30. Results : The limit-of-detection of the Hecin test kit was around 500 dC/mL for the N and ORF1ab targets. Sensitivity and specificity of the Hecin test kit were 98.1% (95% CI: 93.4–99.8%) and 100% (98.1–100%), respectively, when measured against the reference method. The Hecin test kit showed fair sensitivity (80%) in low-positive NPS samples pooled in ratios of 1:5 and 1:10. Its performance in pooled samples could be dramatically improved by adjusting the assay Ct cutoff. Conclusion : The Hecin test kit enables sensitive and specific detection of SARS-CoV-2 in clinical samples and pooled samples.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Comparative evaluation of a dual-target real-time RT-PCR assay for COVID-19 diagnosis and assessment of performance in pooled saliva and nasopharyngeal swab samples

Yip

Leung

et al. 2021

Expert Review of Molecular Diagnostics

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“…One study attempted to deduce the specific patterns among SARS-CoV-2 isolates and accordingly primers were design targeted to nsp2 gene and further use for diagnosis of probe free real-time RT-PCR. Sensitive and rapid SARS-CoV-2-specific real-time RT-PCR assay COVID-19-nsp2 has therefore been developed [69]. In one research, the efficiency of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S) and nucleocapsid (N) SARS-CoV-2 genes was developed and compared.…”

Section: International Statusmentioning

confidence: 99%

Development of RT-PCR Based Diagnosis of SARS-CoV-2

Patankar¹,

Zambare²

2022

Biotechnology to Combat COVID-19

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In the 2020, COVID-19 pandemic disease created an havoc situation world widely and mainly caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). It has been challenging task for researchers, scientists and medico-pharmaceutical organisations to find out rapid and reliable diagnosis methods. Among the all testing services, a Reverse Transcription Polymerase Chain Reaction (RT-PCR) is the more accurate, rapid and authenticated molecular technique used for most of the diagnosis of major diseases. It has been a global priority to fix the rapid diagnosis method to combat against the pandemic COVID-19. Thus, the present chapter mainly focussing on the progress of RT-PCR method development though various processes of data collection on isolation of whole genome sequence, its primer and method designing. In this scenario, India suddenly become the global leader for vaccine development and hence the challenges and RT-PCR kit development in India and rest of the world has been be discussed. World wide many Government and private agencies and industries have taken an initiative for diagnosis of SARS-CoV-2 hence this chapter also summarised the scope of RT-PCR to combat pandemic situation in future.

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“…The virus was found to be a single-stranded (SS) ribonucleic acid (RNA), enveloped virus (Yip et al 2020 ), which invades the upper respiratory tract causing severe infection, and it causes flu-like symptoms like high grade fever, fatigue, dry cough and difficulty in breathing. Also, the chest computed tomography (CT) shows abnormal images.…”

Section: Introductionmentioning

confidence: 99%

Awareness and knowledge of undergraduate dental students about the signs and symptoms of Corona viral infection (COVID-19), and the required infection control measures to prevent its spread

Sharaf

Kabel

2021

Bull Natl Res Cent

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Background Coronavirus disease (COVID-19) is considered a highly contagious disease with flu-like symptoms and causing relatively high level of death. It can be transmitted from a person to another through droplets and that makes the dentists at high risk of infection. Therefore, the aim of the current study was to assess the awareness and knowledge of dental students about the signs and symptoms of Coronavirus disease (COVID-19) and to evaluate their awareness about the required infection control measures during the dental treatment to control the spread of the disease. A questionnaire was formed using Google forms and distributed among dental students and interns in different universities in Cairo, Egypt. Questions were about signs and symptoms of COVID-19, attitude of the dentists toward dental treatment of suspected patients and the required personal protective equipment (PPE) and infection control measures at the dental clinic. Results The majority of the participants strongly agreed/agreed that COVID-19 is a highly dangerous disease, Participants reported that the most common symptom is difficulty in breathing (89.4%) followed by fever (84.4%). Face shield was the most recommended personal protective equipment (PPE) during dental treatment (98.6%) followed by disposable gown (96.3%). The majority of participants (84.8%) recommended using 70% ethyl alcohol as the first method to disinfect surfaces in between dental visits, followed by sodium hypochlorite. Conclusions Dental students and interns in Cairo, Egypt, have good knowledge and awareness about COVID-19 and the necessary precautions required to provide adequate dental treatment for the patients during the pandemic COVID-19; however, the importance of infection control should be highlighted for both clinical and preclinical dental students, to provide safe dental treatment to the patients as well as protection of the dentists and healthcare workers.

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

Cited by 25 publications

References 35 publications

Comparative evaluation of a dual-target real-time RT-PCR assay for COVID-19 diagnosis and assessment of performance in pooled saliva and nasopharyngeal swab samples

Comparative evaluation of a dual-target real-time RT-PCR assay for COVID-19 diagnosis and assessment of performance in pooled saliva and nasopharyngeal swab samples

Development of RT-PCR Based Diagnosis of SARS-CoV-2

Awareness and knowledge of undergraduate dental students about the signs and symptoms of Corona viral infection (COVID-19), and the required infection control measures to prevent its spread

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