Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay for Highly Sensitive Detection of SARS-CoV-2

Ji, Wang; Cai, Kun; Zhang, Ruiqing; He, Xiaozhou; Shen, Xinxin; Liu, Jun; Xu, Jin; Qiu, Feng; Lei, Wenwen; Wang, JinRong; Li, Xinna; Gao, Yuan; Jiang, Yongzhong; Xu, Wenbo; Ma, Xuejun

doi:10.1021/acs.analchem.0c01884

Cited by 78 publications

(96 citation statements)

References 15 publications

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“…There are several differences between our SARS-CoV-2 STN RT-PCR assays and those reported by Wang et al [13]. First, unlike our assays, these assays required locked nucleic acids in their external primer design and their assays with internal primer sets were commercial ones, which would dramatically increase assay cost.…”

Section: Discussionmentioning

confidence: 94%

“…For the five specimens that tested negative by the non-nested N assay but positive by the other three assays, the median Cp value of these specimens was 36. 13 To further determine the clinical utility of the STN RT-PCR assays, 91 follow-up respiratory specimens and 17 follow-up non-respiratory specimens from patients with laboratory-confirmed COVID-19, which tested negative for SARS-CoV-2 by the non-nested COVID-19-RdRp/Hel assay, were tested. These specimens were collected from the patients with COVID-19 between 2 and 42 days after onset of symptoms.…”

Section: Diagnostic Performance Of the Stn Rt-pcr Assays For Sars-covmentioning

confidence: 99%

“…By incorporation of fluorescent probes, these assays are also compatible with commonly used real-time PCR thermocyclers enabling relative quantitation. Recently, Wang et al has developed SARS-CoV-2 STN RT-PCR assays based on a commercial RT-PCR kit [13], but the primer and probe sequences used were not reported. In this study, we designed novel STN real-time RT-PCR assays capable of highly sensitive detection of SARS-CoV-2 in clinical specimens.…”

Section: Introductionmentioning

confidence: 99%

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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

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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Section: Discussionmentioning

confidence: 94%

Section: Diagnostic Performance Of the Stn Rt-pcr Assays For Sars-covmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

Yip

Sridhar

Leung

et al. 2020

IJMS

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“…In the early days of the outbreak, the detection of SARS-CoV-2 by nested RT-PCR method has been Table 2 RT-PCR tests/primers for SARS-CoV-2 in different institutions. In addition, 7 samples with qRT-PCR-positive in the gray zone were confirmed to positive by OSN-qRT-PCR [52]. Compared with the qRT-PCR kit, nested RT-PCR analysis showed higher sensitivity and specificity, indicating that it is more suitable for clinical application to detect SARS-CoV-2 in cases with low viral load.…”

Section: Nested Rt-pcrmentioning

confidence: 98%

Laboratory diagnosis of coronavirus disease-2019 (COVID-19)

Zhao

Bao

et al. 2020

Clinica Chimica Acta

132

116

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The outbreak of Coronavirus Disease-2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has threatened health worldwide. As of the end of 2020, there were nearly 10 million confirmed cases and nearly 5 million deaths associated with COVID-19. Rapid and early laboratory diagnosis of COVID-19 is the main focus of treatment and control. Molecular tests are the basis for confirmation of COVID-19, but serological tests for SARS-CoV-2 are widely available and play an increasingly important role in understanding the epidemiology of the virus and in identifying populations at higher risk for infection. Pointof-care tests have the advantage of rapid, accurate, portable, low cost and non-specific device requirements, which provide great help for disease diagnosis and detection. This review will discuss the performance of different laboratory diagnostic tests and platforms, as well as suitable clinical samples for testing, and related biosafety protection. This review shall guide for the diagnosis of COVID-19 caused by SARS-CoV-2.

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“…The use of two pairs of oligonucleotides allows for a higher number of cycles to be performed, thereby increasing the sensitivity of the PCR. Feng ZS et al previously developed a novel locked nucleic acid (LNA)-based one-step single-tube nested (OSN)-qRT-PCR strategy to detect viral and bacterial pathogens with higher sensitivity and speci city than qRT-PCR and without the need of lid opening [20]. To improve the diagnostic accuracy of nucleic acid detection of SARS-CoV-2 in low viral load samples, they developed and evaluated the sensitivity and accuracy of OSN-qRT-PCR in detecting SARS-CoV-2 [21] .…”

Section: Droplet Digital Pcr (Ddpcr) Is a Third Generation Pcr Basedmentioning

confidence: 99%

Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

Zhang

Dai

Wang

et al. 2020

Preprint

Self Cite

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Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30–60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay for Highly Sensitive Detection of SARS-CoV-2

Cited by 78 publications

References 15 publications

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

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

Laboratory diagnosis of coronavirus disease-2019 (COVID-19)

Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

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