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Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay.
J Virol Methods. 2020 10; 284:113926.JV

Abstract

BACKGROUND

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in the city of Wuhan, Hubei Province, China, has spread worldwide and is threatening human life. The detection of SARS-CoV-2 is critical for preventing new outbreaks, curbing disease spread, and managing patients. Currently, a reverse-transcription polymerase chain reaction (RT-PCR) assay is used to detect the virus in clinical laboratories. However, although this assay is considered to have high specificity, its sensitivity is reportedly as low as 60-70 %. Improved sensitivity is, therefore, urgently required.

METHODS

We used the primers and single-quencher probes recommended by the CDC (N1, N2 and N3) in the USA and the NIID (N1 and N2) in Japan. In addition, we designed double-quencher probes according to the virus sequence provided by the NIID to develop a further assay (termed the YCH assay [N1 and N2]). Using these assays, we conducted RT-PCR with serially diluted DNA positive controls to assess and compare the detection sensitivity of the three assays. Furthermore, 66 nasopharyngeal swabs were tested to determine the diagnostic performances.

RESULTS

The threshold cycle (Ct) value of the RT-PCR was relatively low for the CDC and YCH assays compared with the NIID assay. Serial dilution assays showed that both the CDC and YCH assays could detect low copy numbers of the DNA positive control. The background fluorescence signal at the baseline was lower for the YCH assay compared with the NIID assay. We assessed the diagnostic performance between single- (NIID) and double-quencher (YCH) probes using 66 nasopharyngeal swabs. When the results of YCH-N2 assay were used as a reference, each assay detected SARS-CoV-2 with positive percent agreements of 56 % for NIID-N1, 61 % for YCH-N1, and 94 % for NIID-N2, and 100 % negative percent agreements for NIID-N1, YCH-N1 and NIID-N2.

CONCLUSION

Double-quencher probes decreased the background fluorescence and improved the detection sensitivity of RT-PCR for SARS-CoV-2.

Authors+Show Affiliations

Genome Analysis Center, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan. Electronic address: hirotsu-bdyu@ych.pref.yamanashi.jp.Genome Analysis Center, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan; Department of Gastroenterology, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan.Department of Gastroenterology, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi, Japan; The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

32650037

Citation

Hirotsu, Yosuke, et al. "Double-quencher Probes Improve Detection Sensitivity Toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Reverse-transcription Polymerase Chain Reaction (RT-PCR) Assay." Journal of Virological Methods, vol. 284, 2020, p. 113926.
Hirotsu Y, Mochizuki H, Omata M. Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay. J Virol Methods. 2020;284:113926.
Hirotsu, Y., Mochizuki, H., & Omata, M. (2020). Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay. Journal of Virological Methods, 284, 113926. https://doi.org/10.1016/j.jviromet.2020.113926
Hirotsu Y, Mochizuki H, Omata M. Double-quencher Probes Improve Detection Sensitivity Toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Reverse-transcription Polymerase Chain Reaction (RT-PCR) Assay. J Virol Methods. 2020;284:113926. PubMed PMID: 32650037.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay. AU - Hirotsu,Yosuke, AU - Mochizuki,Hitoshi, AU - Omata,Masao, Y1 - 2020/07/07/ PY - 2020/03/31/received PY - 2020/06/11/revised PY - 2020/07/04/accepted PY - 2020/7/11/pubmed PY - 2020/9/10/medline PY - 2020/7/11/entrez KW - 2019-nCoV KW - COVID-19 KW - Coronavirus KW - Diagnosis KW - Quencher KW - RT-PCR KW - SARS-CoV-2 SP - 113926 EP - 113926 JF - Journal of virological methods JO - J Virol Methods VL - 284 N2 - BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in the city of Wuhan, Hubei Province, China, has spread worldwide and is threatening human life. The detection of SARS-CoV-2 is critical for preventing new outbreaks, curbing disease spread, and managing patients. Currently, a reverse-transcription polymerase chain reaction (RT-PCR) assay is used to detect the virus in clinical laboratories. However, although this assay is considered to have high specificity, its sensitivity is reportedly as low as 60-70 %. Improved sensitivity is, therefore, urgently required. METHODS: We used the primers and single-quencher probes recommended by the CDC (N1, N2 and N3) in the USA and the NIID (N1 and N2) in Japan. In addition, we designed double-quencher probes according to the virus sequence provided by the NIID to develop a further assay (termed the YCH assay [N1 and N2]). Using these assays, we conducted RT-PCR with serially diluted DNA positive controls to assess and compare the detection sensitivity of the three assays. Furthermore, 66 nasopharyngeal swabs were tested to determine the diagnostic performances. RESULTS: The threshold cycle (Ct) value of the RT-PCR was relatively low for the CDC and YCH assays compared with the NIID assay. Serial dilution assays showed that both the CDC and YCH assays could detect low copy numbers of the DNA positive control. The background fluorescence signal at the baseline was lower for the YCH assay compared with the NIID assay. We assessed the diagnostic performance between single- (NIID) and double-quencher (YCH) probes using 66 nasopharyngeal swabs. When the results of YCH-N2 assay were used as a reference, each assay detected SARS-CoV-2 with positive percent agreements of 56 % for NIID-N1, 61 % for YCH-N1, and 94 % for NIID-N2, and 100 % negative percent agreements for NIID-N1, YCH-N1 and NIID-N2. CONCLUSION: Double-quencher probes decreased the background fluorescence and improved the detection sensitivity of RT-PCR for SARS-CoV-2. SN - 1879-0984 UR - https://www.unboundmedicine.com/medline/citation/32650037/Double_quencher_probes_improve_detection_sensitivity_toward_Severe_Acute_Respiratory_Syndrome_Coronavirus_2__SARS_CoV_2__in_a_reverse_transcription_polymerase_chain_reaction__RT_PCR__assay_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0166-0934(20)30178-6 DB - PRIME DP - Unbound Medicine ER -