Establishment of a Nucleic Acid Detection Method for Norovirus GII.2 Genotype Based on RT-RPA and CRISPR/Cas12a-LFS

Human noroviruses are a significant cause of acute gastroenteritis worldwide (Tarr et al. 2021), affecting individuals of all age groups, with young children and the elderly being particularly susceptible (Kapikian et al. 1972; Katayama et al. 2014; Bernstein et al. 2015). It is estimated that noroviruses are responsible for approximately 18 percent of all cases of acute gastroenteritis (Ahmed et al. 2014). Currently, norovirus-induced infectious gastroenteritis is recognized as a leading cause of escalating morbidity and mortality, especially among young children in developing nations (Bok and Green 2012; Lian et al. 2019; Liao et al. 2021). Noroviruses belong to the family Caliciviridae and are characterized as non-enveloped entities with a positive-sense RNA genome approximately 7,500 nucleotides in length, exhibiting variations among different isolates (Lambden et al. 1993; Zhou et al. 2021). The norovirus genome comprises three open reading frames (ORF): ORF1, ORF2, and ORF3, which may synthesize eight proteins (Kobayashi et al. 2016). ORF1 encodes a polyprotein precursor that is proteolytically processed to yield six nonstructural proteins, including RNA-dependent RNA polymerase (RdRp), while ORF2 and ORF3 encode the major and minor structural proteins, respectively (Jiang et al. 1993; Glass et al. 2000).

Phylogenetically, noroviruses are classified into 10 genogroups (GI~GX) with a total of 49 genotypes (9 GI, 27 GII, 3 GIII, 2 GIV, 2 GV, 2 GVI, and one genotype each for GVII, GVIII, GIX [formerly GII.15], and GX) (Chhabra et al. 2019). Moreover, the nucleotide diversity of the RdRp gene, noroviruses can be categorized into 60 P-types (14 GI, 37 GII, 2 GIII, 1 GIV, 2 GV, 2 GVI, 1 GVII, and 1 GX), 2 tentative P-groups and 14 tentative P-types (Chhabra et al. 2019). Five norovirus genogroups, including GI, GII, GIV, GVIII, and GIX, can infect humans (Winder et al. 2022). Among these, norovirus GII is the primary causative agent in gastroenteritis outbreaks, with a single genotype (GII.4) predominating globally (Winder et al. 2022). However, despite over two decades of GII.4 predominance, there has been a recent emergence and increase in the incidence of GII.2 norovirus outbreaks in various countries (Ao et al. 2017; Niendorf et al. 2017).

The primary clinical symptoms of norovirus-induced acute gastroenteritis closely resemble those caused by rotavirus, sapovirus, and astrovirus, primarily presenting as vomiting and diarrhea (Pang et al. 2000; Rockx et al. 2002; O’Ryan et al. 2010; Wikswo et al. 2013). Relying solely on clinical presentation to diagnose norovirus has proven challenging (Bányai et al. 2018; Luo et al. 2019). Additionally, distinguishing norovirus gastroenteritis from bacterial gastroenteritis based solely on clinical manifestations is often difficult (Lively et al. 2018). Therefore, prompt and accurate diagnosis of norovirus infection in food-contaminated samples is crucial for determining appropriate treatment, avoiding unnecessary antibiotic usage, and mitigating the transmission of norovirus gastroenteritis.

Various detection methods for norovirus have been utilized, encompassing both immunological and molecular biological techniques (Wang et al. 2021). While enzyme immunoassays serve as accessible tests, quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered the gold standard for norovirus detection. Both methods utilize stool or emesis samples to detect noroviruses. However, qRT-PCR for norovirus testing encounters certain limitations. For instance, qRT-PCR is time-consuming and complex, requiring specialized skills and equipment, which hampers rapid detection in resource-limited settings. Additionally, the current qRT-PCR cannot differentiate norovirus genotypes in infected samples, potentially overlooking newly emerging variants. Therefore, there is a critical need for methods that can quickly and simply identify norovirus GII.2 genotype strains. Such a method would aid in understanding diversification and gaining insights into the prevalence of norovirus genotype strains among norovirus outbreaks.

In the present study, we developed a rapid and sensitive detection method for the norovirus GII.2 genotype by combining reverse transcription recombinase polymerase isothermal amplification (RT-RPA) with CRISPR/Cas12a technology. This synergistic approach enabled the detection of target RNA at a concentration as low as 10 copies/μl within 35 minutes. The assay results were easily interpretable using lateral flow strips (LFS), enhancing its feasibility for point-of-care applications.

Optimal reaction primers for RT-RPA. This study designed four amplification RT-RPA primers, and five primer combinations were used to determine the optimal RT-RPA primer pairs. All five pairs of primer combinations successfully amplified their corresponding fragments. The amplicon of NoV-GII.2-F1/NoV-GII.2-R2 exhibited specificity and the highest brightness (Fig. 1a). Upon further analysis of the Cas12a-LFS assay results, it was observed that the intensity of the T-line for the NoV-GII.2-F1/NoV-GII.2-R2 exceeded that of the other four (Fig. 1b). Consequently, the NoV-GII.2-F1/NoV-GII.2-R2 primer combination was selected as the optimal primer set for subsequent experiments.

Fig. 1.

Optimum reaction concentration of RT-RPA primer. The concentration of NoV-GII.2-F1/NoV-GII.2-R2 was varied from 0.1 μM to 0.7 μM, with an additional control of 0 μM. As depicted in Fig. 2a and Fig. 2b, a distinct agarose band became visible when the concentration of NoV-GII.2-F1/NoV-GII.2-R2 reached 0.2 μM. Similarly, a pronounced color development on the LFS was observed, indicating a successful reaction with favorable outcomes. Consequently, the optimal primer reaction concentration of NoV-GII.2-F1/NoV-GII.2-R2 was 0.2 μM.

Fig. 2.

Optimal reaction time of RT-RPA for norovirus GII.2. As depicted in Fig. 3, the reaction temperature was varied over 20, 25, 30, 35, and 40-minute intervals. Notably, the reaction achieved excellence when the duration reached 25 minutes. Consequently, the optimal reaction duration was determined to be 25 minutes.

Fig. 3.

Specificity of RT-RPA-Cas12a-LFS for norovirus GII.2. The RT-RPA reaction was performed using nucleic acids from various pathogens as templates. As shown in Fig. 4, the amplification product based on the standard RNA targeting norovirus GII.2 subtype displayed distinct bands on the agarose gel and the LFS. In contrast, the agarose electrophoresis of other viral nucleic acid samples, including RV, AdV, AstV, CV, SaV, BoV, and ddH₂O, did not produce the expected bands, nor did they show the color development on the LFS. These observations suggest that the established RT-RPA-Cas12a-LFS method demonstrates high specificity.

Fig. 4.

Sensitivity of RT-RPA-Cas12a-LFS method for norovirus GII.2. The standard RNA of norovirus GII.2 subtype conserved sequence with different copy numbers was used as a template. As shown in Fig. 5, the clarity of the agarose electrophoresis bands sequentially decreased, reflecting the trend observed in the color development on the LFS. The determined detection limit was as low as 10 copies/μl, indicating a high detection sensitivity level for the RT-RPA-Cas12a-LFS method.

Fig. 5.

Specificity and sensitivity of qRT-PCR detection method for norovirus GII.2. The qRT-PCR reaction utilized nucleic acids from RV, AdV, AstV, CV, SaV, BoV, and NC as templates. As depicted in Fig. 6a, the qRT-PCR amplification fluorescence curve derived from the standard RNA of norovirus GII.2 uniquely crossed the threshold. These results indicate that the developed qRT-PCR method exhibits high specificity, with no cross-reactivity with other viruses. Probit regression analysis established the limit of detection of qRT-PCR for norovirus GII.2 at 10 copies/reaction (95% CI 16.5–31.1), showcasing a high level of detection sensitivity for the qRT-PCR method (Fig. 6b).

Fig. 6.

Analysis of food-contaminated samples. The RT-RPA-Cas12a-LFS method and qRT-PCR method were utilized to detect 60 simulated samples, and representative results are presented in Fig. 7. The RT-RPA-Cas12a-LFS method identified 38 positive samples of norovirus GII.2 and 22 negative samples, whereas the qRT-PCR method detected 39 positive samples of norovirus GII.2 and 21 negatives. Table II shows that the positive consistency rate between the RT-RPA-Cas12a-LFS method and the qRT-PCR method was 100%, the negative consistency rate was 95.45%, and the total coincidence rate was 98.33%. Cohen’s kappa ( κ ) analysis indicated excellent agreement between the two methods, with a κ-value of 0.9638 and a p-value of 0.017 using the Diagnostic Test in the OpenEpi software (www.openepi. com). These findings suggest that the RT-RPA-Cas12a-LFS assay investigated in this study is a valuable tool for the rapid, convenient, and reliable detection of norovirus GII.2 during field inspections.

Fig. 7.

Globally, noroviruses are a leading cause of sporadic cases and acute gastroenteritis (AGE) outbreaks across diverse settings and age demographics (Hassan and Baldridge 2019). Noroviruses GII.4 have maintained their status as the predominant cause of norovirus-associated AGE for nearly three decades. However, an increase in the number of norovirus outbreaks, particularly involving the GII.2 genotype, was reported in various cities in Guangdong during November and December 2016 (Lu et al. 2017), as documented by the provincial surveillance network. Additionally, out of the 21 outbreaks recorded across 10 cities, seventeen (81%) were identified as GII.2, contributing to 760 clinical cases (Lu et al. 2017). In this context, there remains a pressing need for a fast and straightforward norovirus GII.2 detection method. Therefore, the aim of this study is to establish a rapid and simple detection method for norovirus GII.2, particularly in resource-limited areas.

In this study, the development of the RT-RPA--Cas12a-LFS assay for detecting norovirus GII.2 began with the identification of the target sequence within the VP2 region of the norovirus GII.2 genotype. Subse-quently, an optimized protocol was established, starting with the design of RT-RPA primers and crRNA. Various factors affecting RT-RPA amplification efficiency, such as primer sequence, concentration, and reaction time, were evaluated. To enhance sensitivity and specificity while minimizing off-target background noise, we optimized these RT-RPA parameters to obtain optimal reaction efficiency.

Furthermore, the detection of the norovirus GII.2 genotype can be achieved within 25–35 minutes, comprising 20–25 minutes for RT-RPA amplification of the target sequence and an additional 5–10 minutes for the trans-cleavage reaction. This represents a faster process compared to the RT-PCR method. Our RT-RPA-Cas12a-LFS assay achieved a lower detection threshold, detecting as few as 10 copies of target RNA per reaction. Previous studies have indicated that the detection limit of RPA-Cas12a-mediated assays for various pathogens typically ranges from 0.1 to 10 copies/ μl (Nguyen et al. 2022; Qian et al. 2022; Li et al. 2023; Liu et al. 2024). The detection limit is dependent on the optimization of reaction conditions and influenced by the result-readout methods (Qian et al. 2021; Qian et al. 2022; Li et al. 2023). Furthermore, to evaluate the efficacy of our RT-RPA-Cas12a-LFS assay on food-contaminated samples, we tested 60 oyster digestive gland tissues that were contaminated and used for validation. The developed RT-RPA-Cas12a-LFS assay demonstrated remarkable consistency with the qRT-PCR method, boasting a positive predictive agreement of 100%, a negative predictive agreement of 95.45%, and a total coincidence rate of 98.33% specifically for the norovirus GII.2 genotype.

In summary, we have successfully developed an RT-RPA amplification coupled with a CRISPR/Cas12a-LFS detection system for the simple and rapid identification of the norovirus GII.2 genotype, achieving a detection threshold as low as 10 -copies/μl. This assay can be completed within 35 minutes and demonstrates high specificity to the norovirus GII.2 genotype. Importantly, this highlights the potential application of the RT-RPA-Cas12a-LFS technique for norovirus GII.2 detection, offering a highly promising tool for on-site detection, particularly in resource-limited or constrained settings.