A New Auto-RPA-Fluorescence Detection Platform for SARS-CoV-2

In This Article

Abstract and Introduction

Abstract

Objective: The outbreak of COVID-19 caused by SARS-CoV-2 has led to a serious worldwide pandemic. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR)–based methods were recommended for routine detection of SARS-CoV-2 RNA. Because the reaction time and analytical sensitivity of qRT-PCR limits the diagnosis of SARS-CoV-2, development of a quick process of SARS-CoV-2 detection technology with high analytical sensitivity remains urgent.

Methods: We combined isothermal amplification and fluorescence detection technology to develop a new auto-recombinase polymerase amplification (RPA)-fluorescence platform that could be used in the diagnosis of SARS-CoV-2.

Results: By optimization of primers and probes, the RPA platform could detect SARS-CoV-2 nucleotides within 15 min. The limits of detection and specificity of the auto-RPA-fluorescence platform were 5 copies/μL and 100%, respectively. The accuracy of detection of the auto-RPA-fluorescence platform in the 16 positive samples was 100%.

Conclusion: The RPA platform is a potential technology for the diagnosis of SARS-CoV-2 infection.

Introduction

Since the outbreak of COVID-19, caused by SARS-CoV-2, this infectious disease has become a global pandemic that affects many countries in the world. According to the World Health Organization homepage (https://www.who.int), more than 500 million people have been infected with COVID-19, and more than 6 million people have died as of April 2022. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) is a common approach for the detection of SARS-CoV-2 infection. It is routinely performed worldwide, including in the Centers for Disease Control and Prevention (CDC) and other clinical laboratories.^[1,2] Globally, qRT-PCR has been defined as a standard method for the detection of COVID-19. However, this method is time-consuming (the process takes more than 2 h), and requires high purity samples, sophisticated equipment, and well-trained personnel, which can delay the diagnosis of COVID-19. In addition, this method has relatively low analytical sensitivity and poor-quality nucleotide extraction, which causes false-negative results.^[3,4] Therefore, more rapid, sensitive, and accurate diagnostic methods are greatly needed to deal with the COVID-19 pandemic.

To detect COVID-19 infection rapidly, scientists have made various efforts to develop a series of novel detection methods with great potential.^[5] For example, loop-mediated isothermal amplification (LAMP) starts amplification by using Bst DNA polymerase at a constant temperature of approximately 65°C. The LAMP can cycle, elongate, and continue subsequent rounds of amplification with 6 specially designed primers and has high reaction efficiency. The reaction generates magnesium pyrophosphate and results in color change, which can be judged by the naked eye under blue light.^[6,7] However, such a method is mediated by detecting the by-products of loop amplification, which may cause false-positive results due to nonspecific reactions.^[8–11] Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease-based methods have properties that can be used for ultrasensitive diagnostic tests. The Cas nucleases, such as RNA-guided RNase Cas13a and DNase Cas12a, cause collateral cleavage when activated by the recognition of target nucleotide sequences.^[5] Recently, researchers have developed exciting (CRISPR)-based methods by integrating PCR, LAMP, or recombinase polymerase amplification (RPA) with Cas-mediated collateral cleavage,^{[6,9,12–15]} which were amplified by diverse reaction systems.^[5] However, current CRISPR strategies have some disadvantages, including multiple-step operations and long incubation times. A new approach that is more rapid, accurate, and simple for detecting SARS-CoV-2 is urgently needed.

To achieve more rapid and accurate detection of SARS-CoV-2 infection, we applied RPA and a fluorescence detection system to develop an auto-RPA–fluorescence assay for detecting SARS-CoV-2 RNA. The results show that the new assay described here is more rapid and simple than the standard qRT-PCR used currently to detect SARS-CoV-2 and has potential to detect other major pathogenic microorganisms as well.

Table 1. Primers and Probes Used in the Auto-RPA–Fluorescence Platform ^a
Primer/Probe	Sequence 5'→ 3'
N256For	ACTACCGAAGAGCTACCAGACGAAT
N540Rev	CGTGATGAGGAACGAGAAGAGGCTTG
N540For	CAAGCCTCTTCTCGTTCCTCATCAC
N689Rev	GCCTTTACCAGACATTTTGCTCTCA
N860For	GGACCAGGAACTAATCAGACAAGGAA
N1167Rev	GCAGCAGGAAGAAGAGTCACAGTTTG
S3053For	CAGAGCTTCTGCTAATCTTGCTGCTA
S3202Rev	TTGTGAAGTTCTTTTCTTGTGCAGGG
S2531For	TGCTGCTAGAGACCTCATTTGTGCAC
S2850Rev	AAAGCTTGTGCATTTTGGTTGACCAC
Probe N645 exo	GGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCA
Probe N408 exo	GAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAAT
Probe S2560 exo	AACGGCCTTACTGTTTTGCCACCTTTGTCACAGATGAAATGA
Probe S2760 exo	CTATTGGCAAAATTCAAGACTCATTTCTTCCACAGCAA
Probe S3135 exo	GCTATCATCTTATGTCCTTCCCTCGTCAGCACCTCATGG

^aPrimers and probes were designed and selected by the online tool PrimedRPA (see Materials and Methods). All primers and probes were tested by BLAST to detect specificity for SARS-CoV-2 virus genome and subsequently tested in the auto-RPA-fluorescence platform.