Evaluation of the commercially available LightMix® Modular E-gene kit using clinical and proficiency testing specimens for SARS-CoV-2 detection

Yip, Cyril C. Y.; Sridhar, Siddharth; Cheng, Andrew; Leung, Kit Hang; Choi, Garnet Kwan Yue; Chen, Jonathan Hon-Kwan; Poon, Rosana W.S.; Chan, Kwok Hung; Wu, Alan; Chan, Helen Shuk Ying; Chau, Sandy Ka Yee; Chung, Tom Wai Hin; To, Kelvin Kai-Wang; Tsang, Owen Tak Yin; Hung, Ivan Fan Ngai; Cheng, Vcc; Yuen, Kwok Yung; Chan, Jasper Fuk Woo

doi:10.1016/j.jcv.2020.104476

Cited by 52 publications

(53 citation statements)

References 32 publications

(34 reference statements)

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“…This study included archived respiratory specimens from COVID-2019 patients with laboratory confirmation by real-time reverse transcription-polymerase chain reaction (RT-PCR) targeting the E or RdRp-Hel gene as described previously [ 12 , 13 ]. The respiratory specimens included nasopharyngeal swab, saliva, endotracheal aspirate, and sputum.…”

Section: Methodsmentioning

confidence: 99%

Intra-host non-synonymous diversity at a neutralizing antibody epitope of SARS-CoV-2 spike protein N-terminal domain

Kok

Chan

et al. 2021

Clinical Microbiology and Infection

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Objectives SARS-CoV-2 has evolved rapidly into several genetic clusters. However, data on mutations during the course of infection are scarce. This study aims to determine viral genome diversity in serial samples of COVID-19 patients. Methods Targeted deep sequencing of spike gene was performed on serial respiratory specimens from COVID-19 patients using nanopore and Illumina sequencing. Sanger sequencing was then performed to confirm the single nucleotide polymorphisms. Results A total of 28 serial respiratory specimens from 12 patients were successfully sequenced using nanopore and Illumina sequencing. A 75-year-old patient with severe disease had a mutation, G22017T, identified in the second specimen. The frequency of G22017T increased from ≤5% (nanopore: 3.8%; Illumina: 5%) from first respiratory tract specimen (sputum) to ≥60% (nanopore: 67.7%; Illumina: 60.4%) in the second specimen (saliva; collected 2 days after the 1 st specimen). The difference in G22017T frequency was also confirmed by Sanger sequencing. G22017T corresponds to W152L amino acid mutation in the spike protein which was only found in <0.03% of the sequences deposited into a public database. Spike amino acid residue 152 is located within the N-terminal domain, which mediates the binding of a neutralizing antibody. Conclusions A spike protein amino acid mutation W152L located within a neutralizing epitope has appeared naturally in a patient. Our study demonstrated that monitoring of serial specimens is important in identifying hotspots of mutations, especially those occurring at neutralizing epitopes which may affect the therapeutic efficacy of monoclonal antibodies.

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Section: Methodsmentioning

confidence: 99%

Intra-host non-synonymous diversity at a neutralizing antibody epitope of SARS-CoV-2 spike protein N-terminal domain

Kok

Chan

et al. 2021

Clinical Microbiology and Infection

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“…Specific among 17 respiratory viruses, except SARS-CoV. Similar sensitivity with in-house assays (144/28949.8% (144/289) for RdRp/Hel & 50.5 (146/289) for N, In-house RdRp/Hel and -N assays were specific without the interference of SARS-CoV 289 [ 101 ] RT-PCR Cepheids Xpert Xpress kit ( N2,E) In house RdRp, N1, E Cepheids Xpert Xpress 8.26 cp mL −1 NP, Nasal wash Specific, three samples containing various concentrations of heat-inactivated SARS-CoV-2 virus tested positive at three laboratories in both the in-house RT-PCR and the GeneXpert. LOD was lower than the company claim (250 cp mL −1 ).…”

Section: Novel Developed Technologies For Sars-cov-2 Detectionmentioning

confidence: 82%

Diagnostic techniques for COVID-19 and new developments

Sheikhzadeh

Eissa

Ismail

et al. 2020

Talanta

127

129

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COVID-19 pandemic is a serious global health issue today due to the rapid human to human transmission of SARS-CoV-2, a new type of coronavirus that causes fatal pneumonia. SARS -CoV-2 has a faster rate of transmission than other coronaviruses such as SARS and MERS and until now there are no approved specific drugs or vaccines for treatment. Thus, early diagnosis is crucial to prevent the extensive spread of the disease. The reverse transcription-polymerase chain reaction (RT-PCR) is the most routinely used method until now to detect SARS-CoV-2 infections. However, several other faster and accurate assays are being developed for the diagnosis of COVID-19 aiming to control the spread of infection through the identification of patients and immediate isolation. In this review, we will discuss the various detection methods of the SARS-CoV-2 virus including the recent developments in immunological assays, amplification techniques as well as biosensors.

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“…To date, primers and probes used in different RT-PCR assays mainly target ORF1ab, S, E, N regions of SARS-CoV-2 [1,14,17,[19][20][21][22][23][24]. Coronaviruses have a high frequency of mutations and recombination [25][26][27], which may result in mismatches with the currently used primers and probes.…”

Section: Discussionmentioning

confidence: 99%

“…For diagnostic performance evaluation, 213 initial clinical specimens including NPA, NPS, throat swab or saliva collected from 213 hospitalized patients (male: female = 112: 101; median age: 48 years; range: 62 days-103 years) with suspected COVID-19 were included for SARS-CoV-2 detection. Some of these specimens were evaluated previously using our in-house RT-PCR assays for SARS-CoV-2 detection [21]. To further determine the clinical utility of our novel STN real-time RT-PCR assays, we included hospitalized patients with sequential respiratory and non-respiratory specimens sent for SARS-CoV-2 detection for whom the first specimen tested positive but subsequent specimens tested negative by our previously described validated COVID-19-RdRp/Hel real-time RT-PCR assay [14].…”

Section: Viruses Clinical Specimens and Proficiency Testing Samples mentioning

confidence: 99%

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

Yip

Sridhar

Leung

et al. 2020

IJMS

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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.

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Evaluation of the commercially available LightMix® Modular E-gene kit using clinical and proficiency testing specimens for SARS-CoV-2 detection

Cited by 52 publications

References 32 publications

Intra-host non-synonymous diversity at a neutralizing antibody epitope of SARS-CoV-2 spike protein N-terminal domain

Intra-host non-synonymous diversity at a neutralizing antibody epitope of SARS-CoV-2 spike protein N-terminal domain

Diagnostic techniques for COVID-19 and new developments

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

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