key: cord-1019072-1m61jghw
authors: Hirotsu, Yosuke; Mochizuki, Hitoshi; Omata, Masao
title: 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
date: 2020-07-07
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2020.113926
sha: 51b7fc81dc9be9a2abf222d2136738b1c310a9d9
doc_id: 1019072
cord_uid: 1m61jghw

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.

In early December 2019, the first cases of pneumonia of unknown origin were suspected in Wuhan, the capital of Hubei, China [1] . This novel humaninfecting coronavirus was tentatively named "2019 novel coronavirus" (2019-nCoV) by the World Health Organization (WHO) and then later renamed as "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV) [2] . As of March 9, 2020, more than 109,577 cases of coronavirus disease 2019 (COVID-19) had been confirmed, including 80,904 cases in China and 28,673 cases outside of China, with 3,809 deaths globally (WHO Situation Report-49). Human-tohuman transmission of the virus accounts for the virus spreading throughout the world [3] [4] [5] [6] [7] [8] .

SARS-CoV-2 belongs to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China [9] . Phylogenetic analysis has indicated that bats may have also been the original host of SARS-CoV-2 [10] . SARS-CoV-2 has a single-stranded, positive-sense RNA genome that is 29,903 base pairs in length (NCBI Reference Sequence: NC_045512.2) [11, 12 ]. An early viral genome sequence J o u r n a l P r e -p r o o f was published to inform and assist public health control, followed by the genome sequencing data of 169 samples deposited in the database curated by the Global Initiative on Sharing All Influenza Data (GISAID) [13] . These data indicated how the SARS-CoV-2 outbreaks had occurred worldwide based on the observed genetic variations.

Human infections with SARS-CoV-2 have become a global health concern. The diagnosis of COVID-19 is of high priority for patient and public health management to minimize spread of the infection. Chest computed tomography (CT) imaging can be used to diagnose patients with COVID-19 [14] . In addition, a reverse-transcription polymerase chain reaction (RT-PCR) has been developed that uses throat swabs or sputum specimens; however, although this assay is considered to have high specificity, its sensitivity is reportedly as low as 60-70% compared to CT examination [15] [16] [17] . Ai et al. showed RT-PCR test was positive at 59% (601/1,014), whereas chest CT was positive at 88% (888/1,014) in Wuhan, China [15] . Fang et al. showed the initial RT-PCR analysis detected at 71% (36/51) in patients who had abnormality at the rate of 98% (50/51) by the chest CT. Even though the initial RT-PCR test J o u r n a l P r e -p r o o f was negative, repeated RT-PCR tests have increased the cumulative positive rate for COVID-19 [18] .

There is significant demand for a SARS-CoV-2 diagnostic with improved sensitivity that can be used for testing suspected cases. WHO documented the interim guidance of laboratory testing, which denotes nucleic acid amplification test (i.e. RT-PCR) should be conducted to COVID-19 suspected cases [19] .

Low sensitivity in RT-PCR can be the result of several factors including insensitivity inherent to the detection method, variation in the types of detection method used, low initial viral load, types of specimen, and improper clinical sampling [18] . The viral load in nasopharyngeal swabs is higher than that in throat swabs as determined by RT-PCR, suggesting that upper respiratory specimens are superior for genetic testing [20] . To date, protocols of RT-PCR assay to detect SARS-CoV-2 are designed in several countries including USA Thailand (National Institute of Health) [21] .

J o u r n a l P r e -p r o o f 8 The USA CDC assay shows positive percent agreement (100%, 13/13 clinical samples) and negative percent agreement (100%, 104/104) using the primers/probe sets targeting the different regions of nucleocapsid (N) gene [21] .

The NIID assay also shows positive percent agreement (100%, 10/10) and negative percent agreement (100%, 15/15) compared to the result of LightMix Modular SARS and Wuhan CoV E-gene assay [22] .

In the present study, we looked at improving assay sensitivity with the use of an improved detection method. We compared the detection sensitivity among the primer/probe sets designed by the NIID in Japan and the CDC in the USA. Both of these assays use single-quencher probes. We further designed an RT-PCR assay using double-quencher probes based on the same SARS-CoV-2 sequence released by the NIID. The findings indicated that the double-quencher probes reduced the background signal and improved the detection sensitivity of RT-PCR for SARS-CoV-2.

We collected 66 nasopharyngeal swabs between March 11 and April 20, Participation in the study by patients was optional.

The CDC has designed an RT-PCR assay for SARS-CoV-2 and Table 1) .

The NIID has also designed an RT-PCR assay and published associated data [23] . We obtained these primer and probe sets from the NIID (hereafter J o u r n a l P r e -p r o o f called the NIID assay). The NIID assay includes two sets of primers, as well as probes containing a 5'-FAM dye and a 3'-TAMRA dye (Table 1) .

We further designed double-quencher probes (purchased from IDT) based on the same primers/probe sequence reported in the NIID protocol (hereafter called the YCH assay). These probes each incorporate a 5′-FAM dye, an internal ZEN quencher, and a 3′-Iowa Black Fluorescent Quencher (IBFQ)

( Table 1 ). The internal ZEN quencher was incorporated between bases 9 and 10 from the 5′ end of the probe. This design decreased the distance between the dye and the quencher and was expected to reduce the background signal and achieve an improved dynamic range. All of the primer/probe sets in the CDC, NIID, and YCH assays target the N gene of SARS-CoV-2. The CDC assay targets three sites along the N gene, and both of the NIID and YCH assays target two sites along this gene.

For the DNA positive control, we purchased the 2019-nCoV_N_Positive Control (IDT, catalog #10006625), which consists of a plasmid containing the complete N gene (1,260 base pairs) of SARS-CoV-2 (Supplemental Table 1 ). We Table 1 ), and this was used to control for non-specific amplification or internal control.

Total nucleic acids were extracted from nasopharyngeal swabs using the MagMax Viral/Pathogen Nucleic Acid Isolation Kit (ThermoFisher Scientific) on automated machine KingFisher Duo Prime as previously described [24, 25] .

Briefly, we added 400 µL of viral transport media, 10 

All three of the CDC, NIID, and YCH assays amplified the N gene of SARS-CoV-2 ( Figure 1 ). The CDC assay targets three sites along this gene (N1, N2, and N3), and the NIID and YCH assays target two sites (N1 and N2) ( Figure   1 ). A previous study showed that the reverse primer for N1 detection in the NIID assay has one mismatch with the sequence in the current database because it was constructed based on another reported sequence (GenBank: MN908947.1) [23] . This mismatch in the reverse primer did not influence the detection sensitivity compared with the perfectly matched reverse primer [23] .

The CDC and NIID assays both use single-quencher probes; however, in J o u r n a l P r e -p r o o f the present study, we designed an RT-PCR assay (the YCH assay) using new probes incorporating double-quencher technology to reduce the background signal. The expected amplicon sizes for the CDC assay are 72, 67, and 72 bp for the N1, N2, and N3 genes, respectively. The expected amplicon sizes for the NIID and YCH assays are 128 bp and 158 bp for the N1 and N2 genes, respectively.

To assess the detection sensitivity of each of the three RT-PCR assays, we conducted assays using the serially diluted positive control (1, 10, 100, 1,000, and 10,000 copies) as the template (Figure 2 ). We set the threshold value to 0.2 for all assays to determine the threshold cycle (Ct). With over 100 copies of the positive control, amplification was observed in all assays. With 10 copies, we could detect amplification signals for CDC-N2, YCH-N1, YCH-N2, and NIID-N2.

None of the three assays could detect one copy of the positive control. There was no non-specific amplification of the human RPP30 control by any of the assays (data not shown). Overall, the YCH assay achieved the lowest Ct value and, hence, the highest sensitivity when using the serially diluted DNA positive control.

We observed that the Ct values obtained for both the N1 and N2 sites with the NIID assay were relatively high compared with those obtained with the YCH assay (Figure 2 ), despite the primer and probe sequences being equivalent.

We Table 2 ).

Amplification signal of the human RPP30 as an internal control was observed in all samples. These results suggested the detection rate of N2 site was higher than that of N1 site.

If the YCH-N2 assay was used as reference, the percent positive agreements were 56% (10/18) for NIID-N1, 61% (11/18) for YCH-N1 and 94% Assessment of the N1 site was originally reported by researchers in Germany. Previous studies showed that the N1 site of the NIID assay is less sensitive when using the RNA positive control [23, 27] . In this study, we also confirmed that the N1 site of the NIID assay has a limit of detection of 10 copies of the DNA positive control. In addition, we observed that the Ct value was high for the N1 site with the NIID assay. We determined the detection sensitivity using clinical samples and observed the rate of positivity of N2 was higher than that of N1 site (Table 2 ) [25] . All positive samples were detected by N1/N2 sites or N2 site only, but not N1 site only.

During the course of the manuscript submission, CDC recommended the removal of CDC-N3 (USA) primers/probe sets in the kit at March 15, 2020 [21] , because CDC-N3 is intended to detect SARS-like coronavirus and is not specific for SARS-CoV-2. The amplification region of CDC-N3 partially overlapped with that of NIID-N1 and YCH-N1. When the amplification signal was detected in only NIID-N1 but not in NIID-N2, we think it is better to repeat test and confirm the reproducibility. Furthermore, we have to carefully interpret whether the RT-PCR specifically detects SARS-CoV-2 and validate the results by orthologous method (e.g. antigen test and RT-PCR with different primers/probe set).

The background fluorescence signal is mainly determined by two factors:

1) Probe length. Shorter probes have lower background fluorescence, as the shorter the distance between the fluorescent dye and the quencher, the greater the quenching performance owing to fluorescence resonance energy transfer (FRET). 2) Probe capacity for self-quenching. Probes that self-quench through the formation of a three-dimensional structure tend to produce lower background fluorescence.

To detect low virus copy numbers in human specimen, the reduction of background signal is important. To this end, we applied the double-quencher system (YCH assay) for detecting SARS-CoV-2 with RT-PCR. As expected, the use of YCH assay with double-quencher probe decreased the background signal at the baseline and led to a greater dynamic range compared to the NIID assay.

Therefore, to improve detection sensitivity, we recommend the use of doublequencher probes for the detection of SARS-CoV-2, especially when targeting the N1 site of NIID assay.

The Japanese government announced on 6 th March 2020 that the use of NIID assay for SARS-CoV-2 detection approved by the Ministry of Health, Labour J o u r n a l P r e -p r o o f and Welfare in Japan and test fee was covered by the National Health Insurance.

Overall, our findings indicate that YCH assays using double-quencher probes will enable us to detect the low viral loads in COVID-19 patients compared to NIID assay in routine clinical practice.

None. 

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