The development of a droplet digital PCR for accurate detection of SARS-CoV- 2 by simultaneous determination of dual gene targets

doi:10.21203/rs.3.rs-2214895/v1

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The development of a droplet digital PCR for accurate detection of SARS-CoV- 2 by simultaneous determination of dual gene targets

https://doi.org/10.21203/rs.3.rs-2214895/v1

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Real-time fluorescent quantitative PCR is considered as the gold standard for diagnosing COVID-19 worldwide now. But RT-PCR which could yield semi-quantitative results only, and the sensitivity of this method is usually not sufficient to discriminate patients in early stages of infection or with a very low viral load. So, the false-negative phenomenon often brings difficulties to epidemic prevention and diagnosis. More accurate and robust determine method are required for accurate SARS-CoV-2 diagnosis. To offer an attractive platform for SARS-CoV-2 RNA quantification, we report a droplet digital PCR technique for efficient, accurate, and quantitative detection of SARS-CoV-2 RNA. Two pairs of primer and two double-quenched probes targeting the region of ORF1ab and N protein coding genes of SARS-CoV-2 were designed to develop ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested by standard cDNAs and clinical specimens. First, we found the optimal annealing temperature was 53.6℃, the optimal primer concentration was 500 nM per reaction. When both probes of ORF1ab and N protein are applied simultaneously, their concentrations are100 nM and 50 nM, respectively. The optimal concentration of SARS-CoV-2 standard cDNA ranged from 1×10³ to 1×10⁴ copies/µL. And under this condition, the trend line for the measured concentration is Y = 0.9978x-0.4435. Further, the ddPCR were approximately 10 folds greater sensitivity than RT-qPCR assay. The established ddPCR assay with both probes showed to be a highly effective, accurate and reliable method for the sensitive detection of SARS-CoV-2. And the ddPCR method is the most potential and valuable method for clinical detection of SARS-CoV-2 virus.

SARS-CoV-2

Droplet digital RT-PCR

Sensitivity

Specificity

Repeatability.

Since the outbreak of coronavirus disease 2019 (COVID-19), a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is identified as the pathogen [1–2]. In this situation, the development of detection method on SARS-CoV-2 and its related technology are also urgent [3–4]. Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect one or more SARS-CoV-2 genomic targets using sequence-specific primers and fluorescent probe, is considered as the gold standard for diagnosing COVID-19 worldwide. The ORF1ab region, which encodes for 16 non-structural proteins (NSP) mainly involved in the replication process, is the most frequent target for exploiting RT-qPCR protocols. For example, the standard protocol for COVID-19 diagnosis is based on ORF1ab region in China’s CDC and Europe [5–7]. Another number of structural proteins such as the spike (S), the envelope (E), and the nucleocapsid (N) protein are also extensively used in worldwide [1]. However, RT-qPCR, used for the SARS-CoV-2 detection, are only semi-quantitative and the sensitivity of this method is usually not sufficient to discriminate patients in early stages of infection or with a very low viral load [8–10]. The accuracy and repeatability of RT-qPCR still do not satisfy the requirements of clinical practice, especially in samples with low abundances of pathogenic RNA or interfered by other sequences, recovered patients without risk of viral shedding [10–11]. Therefore, more sensitive and robust detection methods, especially for low and residual viral load samples, are required for accurate SARS-CoV-2 diagnosis [12].

Digital droplet polymerase chain reaction (ddPCR) can provide absolute quantification of nucleic acid through Poisson statistics after limiting dilution and endpoint PCR, offering a more acurate and sensitive method than RT-qPCR [13–14]. It has been used to identify patients in the early stages of cancer, to profile environmental microorganisms, and to detect human pathogens [15–16]. In recent years, ddPCR has emerged as a promising tool for pathogen identification in infectious diseases. Pierri et al [1] established the real-time RT-qPCR and droplet digital RT-PCR assay based on the exonuclease region of ORF1b and nsp14, and investigated the effect on the detection of SARS-CoV-2 in nasopharyngeal swabs. Majumdar et al [17] detected the SARS-CoV-2 in formalin-fixed paraffin-embedded tissues and validated using droplet digital PCR by quantifying nucleocapsid (N1, N2) target sequences of SARS-CoV-2, and found the positive rate was higher than real-time RT-qPCR. However, to further improve the accuracy of ddPCR by two different fluorescence labeled probes for simultaneous determination of two target gene fragments had not reported. Here, we established a specific and sensitive ddPCR method for COVID-19 diagnosis with dual fluorescence probes which were designed by the sequence of ORF1ab and N protein, respectively. The sensitivity, reliability and reproducibility of ddPCR were evaluated. We also investigated the agreement of ddPCR and RT-qPCR by detecting standard plasmid.

2.1 Materials, plasmids and dummy virus

The positive standards plasmid, containing partial cDNA sequences of ORF1ab and N protein encoding genes, were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) using pUC57 plasmid vector. The concentration of plasmid DNA was measured using Nano Drop 2000 spectrophotometer (Thermo Sci-entific, USA) and recalculated to plasmid copies/µl using an online calculator. The detailed information of primers and probes is shown in Table 1. The commercial synthetic dummy virus of human coronavirus, such as SARS-CoV-2-abEN (M591001), SARS-ORF1a-N (M596001), MERS-abEN (M592001), hCOV-229E-N (M597001), hCOV-HKU1-N (M597002), hCOV-NL63-N (M597003), and hCOV-OC43-N (M597004) were purchased from Sangon Biotech (Shanghai) Co., Ltd., and used for the sensitivity detection of ddPCR assays. And SARS-CoV-2 dummy virus was used as positive control for the detection of clinical samples.

Table 1

The detailed information of primers used in this study.
Name	Sequence(5’-3’)
Forward primer 1 (ORF1ab)	5’-CCCTGTGGGTTTTACACTTAA-3’
Reverse primer 1 (ORF1ab)	5’-ACGATTGTGCATCAGCTGA-3’
Probe 1 (ORF1ab)	5’-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG -BHQ1-3’
Forward primer 2 (N)	5’-GGGGAACTTCTCCTGCTAGAAT-3’
Reverse primer 2 (N)	5’-CAGACATTTTGCTCTCAAGCTG-3’
Probe 2 (N)	5’-HEX-TTGCTGCTGCTTGACAGATT- BHQ1-3’

2.2 Nucleic acid extraction and reverse transcription

The RNAs of all test samples were extracted with Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions, and were reverse transcribed into first-strand cDNA template using the Evo M-MLV RT Premix (Accurate Biology, China). The positive standards plasmid DNA of SARS-CoV-2 was extracted using the Endo Free Plasmid Midi Kit (Cwbiosciences, China) according to the manufacturer’s instructions.

2.3 Droplet digital PCR assay

Two fluorescent probes of FAM and HEX were designed to detect ORF1ab gene and N protein encoding sequences, respectively. The Bio-Rad QX200 Droplet Digital PCR System (Bio-Rad Laboratories, USA) were employed to quantify the copy number of SARS-CoV-2 according to our previous reports [18].

2.4 Real time fluorescence quantitative PCR assay

The real time RT-qPCR analysis for SARS-CoV-2 was performed using the SLAN-96S real-time fluorescence quantitative PCR detection system (Shanghai Hongshi Medical Technology, China) and SYBR RT-PCR kits (TaKaRa, China) according to the manufacturer’s instructions. The standard curve was generated using standard plasmid DNA of 10-fold serial dilutions. The copy number within each sample was calculated using C_q-value of the sample to the standard curve. No template control was consisted in all reactions and every sample was measured in triplicate.

2.5 Comparison the sensitivity of ddPCR and RT-PCR for SARS-CoV-2 quantification

The positive standard plasmids (1 × 10⁹) of SARS-CoV-2 were first serially 10-fold diluted and used to evaluate the sensitivity and accuracy between ddPCR and RT-qPCR systems. The correlation of mean values for each concentration from both the determination was used to assess the consistency between ddPCR and RT-qPCR measurements.

2.6 Specificity and reproducibility of ddPCR assay

To validate the specificity of the ddPCR assay, nucleic acid extracts of dummy virus for human coronavirus, SARS-CoV-2-abEN, SARS-ORF1a-N, MERS-abEN, hCOV-229E-N, hCOV-HKU1-N, hCOV-NL63-N, hCOV-OC43-N, and no template controls (NTC) were tested. Positive standard plasmids of SARS-CoV-2 with serial dilution copy numbers were used to evaluate the robustness and reproducibility of the ddPCR assay. Each sample was tested in triplicate to evaluate intra- and inter-assay reproducibility.

2.7 Testing of clinical samples

SARS-CoV-2 dummy virus was used as positive control for the detection of clinical samples. A total of 200 negative SARS-COV-2 diagnostic throat swab samples of routine examination from healthy participants are obtained from Huaihe Hospital of Henan University (Kaifeng, China). Random concentration between 10 to 10000 copy numbers of SARS-CoV-2 dummy virus was mixed with throat swab samples from 160 participants, which were used as positive samples. Another 40 samples were used as negative control. Then, the total of 200 samples were randomly sorted and tested by the ddPCR and real-time PCR. No template control (NTC) and positive standards plasmids of SARS-CoV-2 were included in each reaction.

2.8 Statistical analysis

All statistical analyses were performed with SPSS 20.0 software (IBM, Chicago, IL). All experiments were independently performed and repeated at least three times for statistical analyses. The investigator was blinded to patients’ clinical data. Linear regression analyses of the real time PCR calibration curves were performed using Microsoft Excel 2010.

3.1 Thermal cycling temperature optimization of ddPCR

The annealing temperature of ddPCR for SARS-CoV-2 detection was optimized at the following temperatures: 60, 59.6, 58.7, 57.1, 55, 53.6, 52.5, and 52℃. And the positive standard plasmids containing ORF1ab and N protein (pUC57-ORF1ab-N concentration 10⁵ copy numbers per reaction mixture) were used as positive control. As shown in Fig. 1, the optimal annealing temperature was 53.6℃. Then, it resulted in the greatest fluorescence amplitude separation of positive from negative droplets in FAM and HEX probe, and also synthetically generated the largest number of amplification products (number of FAM and HEX positive droplets).

3.2 The optimization of primer and probe concentrations

We first set the probe concentrations as 100 nM, then, primers with different concentrations from 125 to 500 nM were assessed. The optimal primer concentration was 250 nM for both ORF1ab (Fig. 2A, 2B) and N protein (Fig. 2C, 2D). Fixing this primer concentration, we found that the optimal probe concentration for ORF1ab was 100 nM. Then, the difference of fluorescence intensity between positive and negative droplet was significant, indicating higher amplification efficiency of ORF1ab (Fig. 3A, 3B). Similarly, we obtain the optimal probe concentration for N protein detection was also 100 nM (Fig. 3C, 3D).

3.3 The linearity and sensitivity analysis of ddPCR

As shown in Fig. 4 (A and B), the optimal concentration of SARS-CoV-2 standard cDNA ranged from 1×10³ to 1×10⁴ copies/µL. When the copy number of positive plasmids was up to 10⁵ or above, the droplet formation was too concentrated and the separation degree was not high, and the droplet formation was less while the plasmids copy number was less than 10³. Therefore, the plasmids with copy number of 10³ were selected for the following experiment. The trend line for the measured concentration is Y = 0.9391X-0.4795, Y = 1.002X-0.8010 for ORF1ab and N protein, respectively. It indicated that high degree of linearity with the starting concentration of the ddPCR assay for detection of SARS-CoV-2 (R² = 0.9944 for ORF1ab, R² = 0.9878 for N) (Fig. 4C, D). As shown in Fig. 4 (E), we found that the minimum detection limit of ddPCR for positive standard plasmids was 10 copies/µL, while the minimum detection limit of commercial RT-qPCR assay kit was about 100 copies /µL, indicating that the sensitivity of ddPCR was 10 times more than that of RT-qPCR.

3.4 Specificity of ddPCR assay

To estimate the specificity of ddPCR assay, the nucleic acid extracts from dummy virus of human coronavirus such as SARS-CoV-2-abEN, SARS-ORF1a-N, MERS-abEN, hCOV-229E-N, hCOV-HKU1-N, hCOV-NL63-N, and hCOV-OC43-N were used as templates. Without exception, no positive fluorescence signal was observed when using SARS-ORF1a-N, MERS-abEN, hCOV-229E-N, hCOV-HKU1-N, hCOV-NL63-N, hCOV-OC43-N and no template controls (NTC) (Fig. 5). And the detection results of SARS-CoV-2 showed positive droplets in both FAM and HEX channel. Therefore, it suggests that the ddPCR assay was highly specific for detection of the SARS-CoV-2.

3.5 Reproducibility and robustness of ddPCR assay

A serial diluted (10⁴ to 10²) positive standard plasmids for SARS-CoV-2 were prepared to evaluate the reproducibility and robustness of ddPCR assay and each experiment was repeated for three times. The coefficients of variation (CV) and concentration were calculated and showed in Table 2. As shown in Table 2, the CVs of the inter-assay ranged between 1.32% and 4.50%, and the intra-assay CVs for concentration ranged between 3.77% and 4.02%, indicating a good reproducibility of the ddPCR assay.

Table 2

Robustness and reproducibility of ddPCR assay. CV, means coefficient of variation.
Consentration of SARS-CoV-2	Inter-assay (copies/µL)				Intra-assay (copies/µL)
Consentration of SARS-CoV-2	The viral load of 3 tests			CV%	The viral load of 3 tests			CV%
1×10²	61.7	67.5	64.3	4.50	60.2	63.4	65.2	4.02
1×10³	611	596	620	1.99	618	646	601	3.66
1×10⁴	5850	5762	5698	1.32	5862	5436	5673	3.77

3.6 Evaluation of ddPCR assay by clinical samples

To evaluate the optimized ddPCR assay in clinical samples, throat swab samples of healthy participants with or without SARS-CoV-2 dummy virus was used. These samples included 160 positive samples with different concentration of SARS-CoV-2 dummy virus and 40 negative samples. The results of RT-PCR and ddPCR assay were no positive amplification in the 40 negative samples. The total test results for 160 positive samples are shown in Table 3. Among the 160 positive samples, 132 positive cases were tested, 10 samples were suspected, and 18 negative cases for SARS-CoV-2, respectively, using RT-qPCR. Conversely, ddPCR detected 139 positive, 11 suspected and 10 negative for SARS-CoV-2. This result demonstrated that ddPCR have a higher sensitivity than real time PCR, which is consistent with our previous detection results of sensitivity. Although both methods are considered to have a high specificity, ddPCR demonstrated a higher true positive rate and sensitivity than RT-qPCR.

Table 3

Comparison of ddPCR and real-time PCR for positive SARS-CoV-2 clinical samples.
		Real time PCR
		Negative	Suspected	Positive	Total
ddPCR	Negative	10	0	0	10
	Suspected	4	7	0	11
	Positive	4	3	132	139
	Total	18	10	132	160

As the COVID-19 outbreak continues, rapid test of viral load measurement can help identify of infection cases and prevent the transmission of virus [19]. Althrough RT-qPCR is the gold standard for the qualitative and quantitative detection of SARS-CoV-2 virus, it is only a semi-quantitative nucleic acid determination method, which is prone to false negative [20–22]. Especially in the early stages of infection, the viral load was below the detection limit of RT-qPCR and undetectable. Such false negative test results are very detrimental to the prevention and control of COVID-19. More efficient and sensitive detection methods of nucleic acid need to be further developed. Compared with RT-qPCR, ddPCR has higher sensitivity and accuracy, and very efficient for independent DNA quantifications [22].

The part of the ddPCR system is a microfluidic device that generates, splits, and stores droplets as independent reactors for amplifying target nucleic acid sequences. Because most droplets involve either single or no target sequence [23], these sequences can be amplified unbiasedly regardless of their original concentration in the sample. First, we optimized the annealing temperature for ddPCR detection of SARS-CoV-2, and found the optimal annealing temperature was 53.6℃ (Fig. 1). Our results found that most primer/probe sets using in the detection of SARS-CoV-2 virus were tolerant of a wide temperature range, and the background signal was very low at 50℃ to 60.5℃ annealing/extension range, but the separation of positive and negative droplet was insufficient at higher temperatures, which were very accordance to the previous reports of Kinloch et al [8]. Further, we optimized the other determination conditions of ddPCR, and found the optimal primer concentrations and the optimal concentration of SARS-CoV-2 standard cDNA. It has high degree of linearity with the starting concentration of the ddPCR assay for detection of SARS-CoV-2 (R² = 0.9985). Thus, this ddPCR method could be used for nucleic acid detection of SARS-CoV-2 virus.

According to previous reports, ddPCR provides absolute quantification through Poisson statistics after limiting dilution and endpoint PCR, offering a more sensitive method than RT-qPCR [13–14]. We found the sensitivity of ddPCR was over 10 times more than that of RT-qPCR (Table 3). The ddPCR is indeed more sensitive than RT-qPCR for detecting the ORF and N genes, although both methods successfully detected the target genes similarly. Compared to the qPCR, ddPCR method had a low coefficient of variation. Low level of nucleic acid (C_q > 29) in samples can be effectively detected by the ddPCR to produce more precise results. Our results are consistent with those reported in several previous papers, though different target segments were used in the assay [9, 24–26]. The qPCR method needs to rely on standard curve for quantification, and often has false negatives [27]. The false negative results may be caused by the poor sampling techniques, which leads to the viral loads in samples varied significantly [28–29]. For another, various PCR inhibitors may be present in the assay samples, thus interfering with the assay results. But the ddPCR, relying on a microfluidic system, can decompose a 20 µL sample into 20000 droplets, thus greatly reducing the influence of PCR interfering agents and improving the accuracy of the assay[30]. Therefore, the ddPCR has more precise and statistically significant results than RT-qPCR, showing an advantage in detecting low levels of pathogenic RNA/DNA in samples.

In clinical sample testing, there were 18 samples that tested negative by RT-qPCR, but 4 of them were positive and 4 of them were suspected by ddPCR. Taken together, these results suggest that the established ddPCR method is a more effective method for the precise quantification of SARS-CoV-2 virus compared with the RT-qPCR system, especially in detecting extremely lower concentrations of SARS-CoV-2. This is very accordance to our previous reports of quantitative detection of porcine reproductive and respiratory syndrome virus [18]. Therefore, the nucleic acid detection assay of ddPCR is the most potential and valuable method for clinical detection of SARS-CoV-2 virus now.

Funding

This work was supported by the Key Science and Technology Fund of Kaifeng in China (NO. 20FK005) which is for the author of Yijie Zhang and Dengke Bao. And another two fundings of Key Program of the Higher Education of Henan Province in China (NO. 212102310821 and NO. 222102310426) for Hongliang Liu and Juanmei Zhang, respectively, also provided partial supports for this work.

Ethics approval

All experiments in this study were performed in accordance with relevant guidelines and regulations and were approved by Ethics Committee of Henan University. The positive virus samples used in this study were the commercial synthetic dummy virus of human coronavirus, which can not cause infection or transmission, and it is no ethical issues involved.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

The data that support the findings of this study are available on reasonable request from the corresponding author.

Code availability

Not applicable.

Competing interests

The authors declare they have no competing interests.

Author Contributions

Juanmei Zhang, Jingru Han and Yixuan Liang designed and carried out the experiment of method determination, drafted and revised the manuscript. Chunyang Bai and Wenhua Liu help to complete the collection and arrangement of clinical samples. Xiaoyan Wang and Ziqian Liao helped to collect and sort out data and experiment pictures. Qingguo Zhu and Jianghao Guo helped to carry out the experiments. Hongliang Liu assisted in the analysis of clinical samples. Dengke Bao led the relevant project and designed the experiment. Yijie Zhang helped design the experiment and provided project support.

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The development of a droplet digital PCR for accurate detection of SARS-CoV- 2 by simultaneous determination of dual gene targets

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Materials, plasmids and dummy virus

2.2 Nucleic acid extraction and reverse transcription

2.3 Droplet digital PCR assay

2.4 Real time fluorescence quantitative PCR assay

2.5 Comparison the sensitivity of ddPCR and RT-PCR for SARS-CoV-2 quantification

2.6 Specificity and reproducibility of ddPCR assay

2.7 Testing of clinical samples

2.8 Statistical analysis

3. Results

3.1 Thermal cycling temperature optimization of ddPCR

3.2 The optimization of primer and probe concentrations

3.3 The linearity and sensitivity analysis of ddPCR

3.4 Specificity of ddPCR assay

3.5 Reproducibility and robustness of ddPCR assay

3.6 Evaluation of ddPCR assay by clinical samples

4. Discussion

Declarations

References

Additional Declarations

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