Rapid and accurate detection of SARS-CoV-2 using the RHAM technology

doi:10.21203/rs.3.rs-3109029/v1

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Rapid and accurate detection of SARS-CoV-2 using the RHAM technology

https://doi.org/10.21203/rs.3.rs-3109029/v1

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published 20 Dec, 2023

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Rapid and sensitive detection of pathogens is of utmost importance in interrupting the transmission chain of infectious diseases. In recent years, this has proven to be vital during the coronavirus disease (COVID-19) global pandemic that put countless lives at risk. Numerous molecular diagnostic methods were used, including RT-PCR, NASBA, E-SDA, E-RCA, LAMP, and RPA. However, these technologies potentially require primer optimization and complex instruments. Here, we propose the RHAM (RNase Hybridization-Assisted amplification) system as a rapid, specific, and sensitive molecular diagnosis platform. Combining the LAMP and RNase HII-mediated fluorescent reporter, the RHAM system can amplify and visualize the target in one isothermal system with high sensitivity (5×10² copies/mL). There was no cross-reactivity with other common respiratory viruses. Analysis of clinical samples revealed the RHAM system to generate positive signals within 15 minutes without false positive or negative results. The present study shows that RHAM is not only an ideal platform for detecting pathogens, such as SARS-CoV-2 but can be potentially applied in POCT settings.

Biological sciences/Biotechnology/Assay systems

Health sciences/Health care/Diagnosis

Infectious diseases continue to be a long-term threat and burden to global public health. During the 20th Century, the influenza virus triggered several epidemics, causing millions of deaths^1,2. In the twenty-first century, the prevalence of coronavirus led to economic recessions in 2003 and 2004, as well as 2020–2022. By May 2023, more than seven million individuals were infected. Other than acute respiratory symptoms and potential health-related effects, infectious viruses also induce chronic genital tract and nasal cavity infections, which may cause cervical and nasopharyngeal carcinoma, leading to greater health consequences to the patient compared to the respiratory pathogen alone³.

Currently, PCR, a molecular biologic technique, remains the gold standard for the identification of most infectious pathogens and is recommended by clinical guidelines due to its high sensitivity and specificity ^4–8. Due to the requirement of well-trained technicians, expensive equipment, and a specific laboratory environment, PCR testing can be somewhat less accessible and its application is limited in most developing countries, as well as in clinical and healthcare centre settings in developed countries without qualified technicians⁹. In contrast to PCR, which requires multiple thermal cycling, isothermal amplification can be performed under a constant temperature without complicated procedures^9–11. Further, equipment costs for isothermal amplification are much lower than that of PCR. Isothermal nucleic acid amplification technology provides a rapid and simple experimental approach compared to the rapid antigen test (RAT); however, its sensitivity and specificity are far superior, making it a promising technology to develop low-cost diagnostic tools ^12,13.

To generate a rapid and easily adaptable molecular diagnostic platform, we developed a new isothermal amplification method termed RHAM (RNase HII-assisted Amplification), which consists of Loop-Mediated Isothermal Amplification (LAMP)-mediated exponential amplification with an RNase HII reporter for signal visualization in a single reaction. In the initial reaction, with the assistance of the conventional LAMP primer set, the target sequence is exponentially amplified by Bst DNA polymerase¹⁴. In the latter stage, the ribonucleotide-containing fluorescent probe, labelled with a fluorophore at the 5’-end and quencher at the 3’-end, then hybridizes with the amplification product, in which the 5’ to the ribonucleotide can be recognized and nicked by RNase HII within the context of DNA-probe duplex. The digested fluorescent probe then dissociates from the targeted amplicon, resulting in the release of the fluorescent group from the quenching group, leading to an increase in the fluorescence signal, which can be read and recorded by a qPCR machine and a positive test result obtained within 10 minutes.

This novel method can be used for the rapid, sensitive, and specific detection of pathogens, such as severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). The present study demonstrates that RHAM can detect the extracted nucleic acid rapidly, sensitively, and specifically, and also directly detect clinical samples without nucleic acid extraction. RHAM provides reliable technical support for the development of a sensitive, rapid, convenient, and portable POCT product and provides a broad prospect for the on-site detection of pathogens.

Development of the RHAM system for detecting SARS-CoV-2 RNA

In the fluorescence-based LAMP detection protocol, reverse transcription of the target genome generates complementary DNA (cDNA). Subsequently, LAMP primers anneal to the cDNA and the cDNA is amplified using Bst DNA polymerase. The resulting product contained is a repeat of the amplicon linked by a single-stranded loop in a long concatemer (Fig. 1A, B)¹⁵. To enhance LAMP detection, RNase HII and the fluorescent probe were added to the LAMP reaction mix for the specific detection of amplicons targeting Orf1ab. During the LAMP process, the ribonucleotide-containing fluorescent probe, labeled with a fluorophore at the 5’-end and a quencher at the 3’-end, can hybridize with the single-stranded LAMP products. Upon recognition by RNase HII, the RNA-DNA hybrid was then cleaved, resulting in the release of the fluorophore (Fig. 1C). We combined the RNase HII, ribonucleotide-containing fluorescent probe with the LAMP system (hereafter named RHAM).

To compare LAMP and RHAM, experiments were performed for the detection of SARS-CoV-2. The real-time kinetics of positive (purified SARS-CoV-2 RNA reference, 100 copies/reaction) and negative samples (no template control) targeting the Orf1ab gene in LAMP reactions were monitored. Positive samples were found to display fluorescence after incubation for 15 min; however, both positive and negative samples showed similar fluorescence intensities after incubation for 27 min, indicating the risk of a high false-positive rate, limiting the contrast between low copy and negative samples (Fig. 2A). To evaluate the performance of RNase HII, a positive fluorescent signal can be observed when adding both the ribonucleotide-containing fluorescent probe and the target single-stranded (ssDNA) compared to the negative control (without ssDNA; Fig. 2B). Next, the RHAM system results showed that the single pot system can robustly detect purified SARS-CoV-2 RNA in less than 10 min without a false positive signal (Fig. 2C) in comparison to the LAMP system (Fig. 1C). Therefore, the RHAM system showed an evident improvement compared to LAMP. The data above demonstrate that RHAM is a more reliable nucleic acid detection system, which can be used to detect SARS-CoV-2 RNA.

RHAM is highly sensitive and specific

RHAM was established successfully for the rapid detection of SARS-CoV-2; however, its performance required further clarification. To assess the sensitivity and specificity of RHAM, its detection limit was examined using SARS-CoV-2 RNA samples. A series of diluted SARS-CoV-2 RNA samples (1×10⁵–5×10² copies/mL) were prepared, with each added to 25 µL of RHAM reaction mix (Fig. 3A). We then monitored the fluorescence of each dilution and calculated their coefficient of variations (CVs). All SARS-CoV-2 RNA positive samples showed fluorescence signals within 25 min; however, the no template control did not display any fluorescence signal (Fig. 3A). The specificity of the primer sets and probe was also analyzed for SARS-CoV-2 diagnostics. Nucleic acids of other respiratory pathogens, including coronavirus (HKU1, OC43, NL63, 229E), SARS coronavirus, MERS coronavirus, influenza A H1N1, H5N1, H7N9 and H9N2, influenza B Virus, respiratory syncytial virus types A and B, human parainfluenza virus type II, adenovirus types 3 and 7, enterovirus EV71, Mycoplasma pneumoniae, Epstein-Barr virus, human cytomegalovirus, and Mycobacterium tuberculosis were synthesized. The extracted nucleic acids were added to the RHAM reaction mix. There was no cross-reactivity with other respiratory pathogens, with only SARS-CoV-2 displaying fluorescent signals at a run time of up to 40 min (Fig. 3B), thus reflecting the specificity of the RHAM system. These data prove that RHAM is a rapid, sensitive, and specific technology for nucleic acid detection.

Fast and accurate detection of SARS-CoV-2 from clinical samples by RHAM without RNA extraction

To assess the ability of RHAM in POCT, the detection of clinical nasopharyngeal samples without RNA extraction was performed. Nasopharyngeal swabs were collected from patients and added to the storage medium. Positive and negative samples were first determined to use commercially available RT-PCR kits. To evaluate the performance of RHAM, 10 SARS-CoV-2 positive and 10 SARS-CoV-2 negative samples were included and randomized. For RHAM detection, 5 µL of the medium was mixed with 5 µL of lysis buffer. The mixture was then added to the RHAM reaction mix to a final total volume of 25 µL (Fig. 4A). Positive results were obtained for all RT-PCR confirmed positive samples using the RHAM platform within 25 min (Fig. 4B). Moreover, all the SARS-CoV-2 negative samples did not demonstrate any fluorescence signals at a run time of up to 40 min (Fig. 4B). The data demonstrate 100% accuracy in SARS-CoV-2 diagnosis using the RHAM platform, indicating that the method is a robust and accurate technology to detect SARS-CoV-2 in clinical applications.

Since the 20th century, the global outbreak of respiratory pathogens, such as influenza A virus and SARS-CoV-2, highlight the critical need for a fast and reliable diagnostic tool to identify infectious agents, which play a key role in controlling epidemics. Given its rapid and easy-to-use technique, RAT is applied largely in COVID-19 POCT as well as in the self-testing setting. However, the low sensitivity at disease onset hinders RAT from becoming an ideal disease-control tool^12,13.

As nucleic acid amplification technology (NAAT) is analogous to PCR, isothermal amplification technology can amplify nucleic acids of the targeted pathogen with specific primers and has a theoretically higher sensitivity than RAT^16,17. Since the amplification process is at a constant temperature, the reaction time is much shorter than that of PCR. Combining these advantages, isothermal amplification becomes a promising candidate to develop a disease-control tool.

Current isothermal nucleic acid amplification techniques, such as nucleic acid sequence-based amplification (NASBA), exponential strand displacement amplification (E-SDA), exponential rolling circle amplification (E-RCA), LAMP, helicase-dependent amplification (HAD), recombinase polymerase amplification (RPA), and exponential amplification reactions (EXPAR) ^18–23, provide rapid and simple experimental procedures for nucleic acid-based diagnosis. Yet, these technologies still have several shortcomings. LAMP and EXPAR have a high risk of non-specific amplification, and it is difficult to comprehensively eliminate false-positive amplicons caused by the accumulation of non-specific products⁹. The RPA system comprises a complex enzyme mix, and it is difficult to couple crude samples to the RPA system. RCA requires a circular template, often requires an additional ligation step, and its application is complicated. We examined the performance of RHAM and LAMP, with RHAM demonstrating rapid and accurate detection of RNA samples over the LAMP system, reflecting a promising platform for isothermal detection and POCT application.

In summary, we here demonstrate the development of a new isothermal amplification technology termed RHAM, which is based on RNase HII and LAMP, to detect targets rapidly and accurately. Further, RHAM can detect clinical samples in 20 min without the need for a laboratory-based environment and extra equipment, demonstrating its tremendous potential to develop a diagnostic tool in PoC or even self-testing areas. However, further clinical samples should be analyzed to additionally demonstrate the sensitivity and specificity of the RHAM system.

Virus samples

MERS coronavirus (ATCC, VR-3248SD), coronavirus OC43 (ATCC, VR-1558D), HKU1 (ATCC, VR-3262SD), 229E (ATCC, VR-740DQ), NL63 (ATCC, VR-3263SD), SARS coronavirus (ATCC, VR-3280SD), influenza A H1N1 (ATCC, VR-1469), H5N1 (ATCC, VR-1647), influenza B Virus (ATCC, VR-1735D), respiratory syncytial virus type A (ATCC, VR-1804DQ) and B (ATCC, 1850DQ), human parainfluenza virus type II (ATCC, VR-92DQ), adenovirus type 3 (ATCC, VR-3DQ) and 7 (ATCC, VR-7DQ), enterovirus EV71 (ATCC, VR-1775DQ), Mycoplasma pneumoniae (ATCC, 29342), human cytomegalovirus (ATCC, VR-1590), and Mycobacterium tuberculosis (ATCC 25177) were purchased from the ATCC SARS-CoV-2 RNA (GBW(E)091098) was purchased from the National Institute of Metrology (China, CNRN). SARS-CoV-2 clinical samples were collected by the KAVI-ICR laboratory in Kenya approved by the ethics committee.

LAMP reaction

The LAMP primer set targeting the Orf1ab gene was designed by PrimerExplorer (http://primerexplorer.jp/) and synthesized by Hippo Bio (Huzhou, China). The RHAM probe was designed using Primer 5.0 and synthesized by Hippo Bio (Huzhou, China). The LAMP reaction contained a final concentration of 1.6 µM FIP/BIP, 0.2 µM F3/B3, and 0.4 µM Loop F/B primers. Ten µmol of Syto9 fluorescent dye (ThermoFisher, S34854), 1X isothermal amplification buffer (20 mM Tris-HCl, 50 mM KCl, 10 mM (NH4)₂SO₄, 2 mM MgSO₄ (Total 8 mM in Reaction), 0.1% Tween 20, pH 8.8), 1.4 mM dNTPs (New England Biolabs N0447), 8 U Bst DNA polymerase, large fragment [Bst DNA Polymerase, Large Fragment (New England Biolabs M0275)], 7.5 U WarmStart RTx Reverse Transcriptase (New England Biolabs M0380), and various templates were included in a 25 µL reaction. The LAMP reaction was performed at 65°C on a qPCR machine (SLAN-96P®, Hongshi, Shang Hai Hongshi Medical Technology Co., Ltd, Shanghai, China) for 40 min, with fluorescence measurements every 60s.

RHAM reaction

RNase HII reporting system signal was verified by adding 5U RNase HII nuclease (New England Biolabs, M0288), 5 pmol probe, and 0.5 pmol complementary chain template to 25 µL of LAMP reaction system, reacting on the SLAN-96P® platform at 65°C for 40 min, with fluorescence measurements performed every 60s. The RHAM reaction was then established in the LAMP reaction, and 5 U RNase HII and 5 pmol probes were added to the 25 µL LAMP reaction. The RHAM reaction was performed at 65°C on the SLAN-96P® platform for 40 min, with fluorescence measurements performed every 60s.

SARS-CoV-2 clinical sample collection and testing

Nasopharyngeal swabs were collected and transported using a virus sampling tube with transport medium (MT0301-2, YOCON Biotechnology Co., Ltd.), which was further submitted to the KAVI-ICR laboratory in Kenya. The RNA was processed by an RNA extraction kit (TIANamp Virus DNA/RNA Kit, DP315, TIANGEN BIOTECH (BEIJING) CO., LTD, Beijing, China). SARS-CoV-2 was determined using the Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (fluorescent PCR) (Liferiver, Shanghai, China), which contained primers and probes for the novel coronavirus (2019-nCoV) ORF1ab, N, and E genes. The three viral targets were qualitatively detected on the SLAN96P® platform. The KAVI Clinical Research Institute in Kenya was commissioned to use SARS-CoV-2 clinical samples to evaluate the performance of the RHAM system on the SLAN-96P® platform.

Ethical declarations

All experimental protocols and clinical samples were approved the ethics committee of KAVI Clinical Research Institute in Kenya. All methods were carried out in accordance with relevant guidelines and regulations. We received written informed consent of all participants.

Ethical declarations

ACKNOWLEDGMENTS

Funding

The present study was funded by the innovation and entrepreneurship leading team project in Guangzhou, China （PN: 202009030009）

Data availability

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

Author contributions

Zhuo Xiao, Xiaoli Liu, and Xiaolong Kang designed the experiments. Xiaoli Liu Xiaolong Kang, Yaoheng Feng, and Lijun Zheng performed the experiments. Xiaoli Liu and Xiaolong Kang supervised the research. All authors discussed the results and commented on the manuscript.

Competing interests

There are no conflicts of interest to declare.

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No competing interests reported.

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You are reading this latest preprint version

Rapid and accurate detection of SARS-CoV-2 using the RHAM technology

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Development of the RHAM system for detecting SARS-CoV-2 RNA

RHAM is highly sensitive and specific

Fast and accurate detection of SARS-CoV-2 from clinical samples by RHAM without RNA extraction

Discussion

Material and Methods

Virus samples

LAMP reaction

RHAM reaction

SARS-CoV-2 clinical sample collection and testing

Ethical declarations

Declarations

Funding

Data availability

Author contributions

Competing interests

References

Additional Declarations

Status:

Journal Publication

Version 1